( PDF ) Caracterização bioquímica, estrutural e funcional de vesículas secretadas por leveduras

Download PDF

ads:

Caracterização bioquímica, estrutural e funcional de vesículas

secretadas por leveduras

Débora Leite de Oliveira

Orientadores: Marcio Lourenço Rodrigues e Leonardo Nimrichter

Rio de Janeiro

Janeiro/ 2011

Tese de Doutorado apresentada ao Programa de

Pós-graduação em Química Biológica, Instituto de

Bioquímica Médica da Universidade Federal do Rio

de Janeiro, como parte dos requisitos necessários à

obtenção de título de Doutor em Ciências (Química

Biológica).

ads:

Livros Grátis

http://www.livrosgratis.com.br

Milhares de livros grátis para download.

Trabalho realizado no Laboratório de Estudos Integrados em

Bioquímica Microbiana, Departamento de Microbiologia

Geral, Instituto de Microbiologia Prof. Paulo de Góes, Centro

de Ciências da Saúde, Universidade Federal do Rio de Janeiro,

sob orientação dos Professores Marcio Lourenço Rodrigues e

Leonardo Nimrichter.

ads:

iii

FICHA CATALOGRÁFICA

Oliveira, Débora Leite

Caracterização bioquímica, estrutural e funcional de vesículas secretadas por leveduras/

Débora Leite de Oliveira – Rio de Janeiro, 2011

X, 149f.

Tese de Doutorado em Ciências (Química Biológica)

Universidade Federal do Rio de Janeiro/ Instituto de Bioquímica Médica, 2011.

Orientadores: Marcio Leorenço Rodrigues e Leonardo Nimrichter

Referências Bibliográficas:

1. Vesículas 2. Secreção não- convencional 3. Cryptococcus neoformans 4. Exossomos

5. Cápsula 6. Saccharomyces cerevisiae

I. Rodrigues, Marcio

II. Nimrichter, Leonardo

III. UFRJ, Instituto de Bioquímica Médica, Doutorado em Ciências (Química Biológica)

IV. Caracterização bioquímica, estrutural e funcional de vesículas secretadas por

leveduras/ Débora

FOLHA DE APROVAÇÃO:

Caracterização bioquímica, estrutural e funcional de vesículas secretadas por leveduras

Débora Leite de Oliveira

Rio de Janeiro, 12 de Janeiro de 2011.

_____________________________________________________________________________

(Dr. Marcio Lourenço Rodrigues, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ)

______________________________________________________________________________

(Dr. Leonardo Nimrichter, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ)

______________________________________________________________________________

(Dra. Débora Foguel, Instituto de Bioquímica Médica, UFRJ)

______________________________________________________________________________

(Dra. Thaís, Instituto de Microbiologia Prof. Paulo de Góes, UFRJ)

______________________________________________________________________________

(Dra. Rosana Puccia, Departamento de Microbiologia, Imunologia e Parasitologia, Unifesp)

Dedico esta tese àquele que esteve sempre

presente, exercendo influência sobre minhas

escolhas profissionais, àquele que simboliza a união

do nosso grupo: a Jorge José Bastos Ferreira.

Agradecimentos

Agradeço pela oportunidade de presenciar um momento de reforma na ciência

nacional, o qual conduzirá a Ciência a lugar de destaque em nossa cultura e sociedade.

Intuitivamente, acredito que as conseqüências sociais desta reforma serão impactantes. É

gratificante saber que a concretização dos meus ideais, aqui colocados nesta tese, participa da

atmosfera de mudança que respiramos hoje no meio científico. Agradeço portanto pela chance

de contribuir com o desenvolvimento científico do Brasil. Obrigada à todos que me ajudaram a

construir esta tese, obrigada sobretudo aos meus orientadores Marcio e Leonardo pela solidez,

cumplicidade e segurança.

Dentre colaboradores, amigos e todos que participaram desta etapa da minha

formação, agradeço francamente a: Allan Guimarães, André Luiz Souza dos Santos e todo o seu

grupo, Arturo Casadevall, Célio Freire de Lima, Ernesto Nakayasu, Geralda Almeida, Igor de

Almeida, José Roberto Meyer Fernandes, Joshua Nosanchuk, Kildare Miranda, Radames

Cordero, Susana Frases, Vivek Malhotra e Vivian Rumjanek. Agradeço especialmente a toda

equipe do Laboratório de Estudos Integrados em Bioquímica Microbiana, por tornarem a minha

rotina ainda mais prazerosa. Acredito que o aprendizado se dá de forma mais contundente

quando acompanhado de experiências positivas, portanto, muito obrigada pelos ensinamentos

e pela diversão! Por fim, agradeço ao Instituto de Bioquímica Médica pelo acolhimento e

excelência.

vii

RESUMO:

Caracterização bioquímica, estrutural e funcional de vesículas secretadas por leveduras

Débora Leite de Oliveira

Orientadores: Marcio Lourenço Rodrigues e Leonardo Nimrichter

Os mecanismos pelos quais macromoléculas são transportadas através da parede celular

em fungos ainda são pouco conhecidos. O Cryptococcus neoformans, agente etiológico da

criptococose, apresenta uma cápsula polissacarídica externa ao corpo celular. Foi demonstrado

nesta tese que o polissacarídeo capsular é exportado para o ambiente extracelular em vesículas

membranosas. A caracterização bioquímica destes compartimentos vesiculares revelou a

presença de diferentes moléculas associadas a virulência, incluindo lipídeos, polissacarídeos e

proteínas. O papel da secreção vesicular durante a interação do fungo com a célula hospedeira

também foi avaliado. Foi demonstrado que macrófagos podem incorporar vesículas

extracelulares produzidas pelo C. neoformans. O tratamento de macrófagos com vesículas

fúngicas resultou na modulação da produção de citocinas e óxido nítrico além de interferir com

a capacidade fagocítica e da atividade microbicida. Estes dados estabeleceram que vesículas

secretadas pelo C. neoformans são biologicamente ativas. Com o intuito de elucidar quais vias

do tráfego intracelular estão potencialmente envolvidas na biogênese vesicular a levedura

Saccharomyces cerevisiae foi usada como organismo modelo. Vesículas foram isoladas de cepas

mutadas em diferentes genes que controlam passos importantes na via secretora clássica e na

via de formação de corpos multivesiculares (MVBs). Análises combinadas por microscopia

eletrônica de transmissão, espalhamento de luz, análises cromatográficas e proteômicas

revelaram que mutações em ambas as vias afetam a composição de vesículas extracelulares,

embora nenhuma das mutações analisadas interrompa a secreção de vesículas. Análises

lipídicas mostraram que mutantes com defeitos na secreção pós-Golgi apresentam uma cinética

mais lenta de liberação de vesículas. Estes dados sugerem que fungos produzem vesículas

extracelulares de atividade biológica de alta relevância, em processos que associam eventos de

secreção convencional e não-convencional.

viii

ABSTRACT:

Biochemical, structural and functional characterization of vesicles secreted by yeast cells

Débora Leite de Oliveira

Orientadores: Marcio Lourenço Rodrigues e Leonardo Nimrichter

The mechanisms by which macromolecules are transported across the cell wall of fungi are

still poorly known. Cryptococcus neoformans, the causative agent of cryptococcosis, has a

polysaccharide capsule outside of the cell body. It was demonstrated in this study that capsular

polysaccharides are exported to the extracellular environment within membrane vesicles. The

biochemical composition of these vesicular compartments revealed the presence of different

molecules associated with virulence, including lipids, polysaccharides and proteins. The role of

vesicular secretion during the interaction of fungi with host cells was also addressed. It was

demonstrated that macrophages can incorporate extracellular vesicles produced by C.

neoformans. Treatment of the macrophages with fungal vesicles resulted in modulation of

cytokine and nitric oxide production, phagocytic ability and microbicidal activity. These findings

establish that cryptococcal vesicles are biologically active. To elucidate what intracellular traffic

pathways originate the extracellular vesicles, the yeast Saccharomyces cerevisiae was used as a

model organism. Vesicles were isolated from cells with mutations in different genes involved in

key steps of the secretory or multivesicular body (MVB) formation pathways. Transmission

electron microscopy in combination with dynamic light scattering, chromatographic approaches

and proteomics revealed that mutations in both pathways affected the composition of yeast

extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that

mutants with defects in Golgi-related components of the classic secretory pathway had slower

vesicle release kinetics. These results suggest that fungi produce extracellular vesicles with

highly relevant biological activities through cellular processes that combine events of both

conventional and non-conventional secretory pathways.

LISTA DE ABREVIATURAS:

ABC (“ATP Binding Cassette”) – cassette de ligação a ATP

AcbA (“acyl-coenzyme A binding protein”) – proteína ligadora de acil- coenzima A

CMH – ceramida monohexose

CMI (“cell mediated immunity”) – imunidade mediada por célula

CNS (“central nervous system”) – sistema nervosa central

COP (“coat protein”) – proteínas de revestimento

CtxB-FITC – subunidade B da toxina do cólera marcada com FITC

CWP (“cell wall protein”) – proteína de parede celular

DAPI (“4',6-diamidino-2-phenylindole”) - 4',6-diamidino-2-fenilindol

DC – célula dendrítica

DiI (“dialkylcarbocyanine iodide”) – iodeto de dialquilcarbocianina

DTT (“1,4-Dithioerythritol”) - 1,4-Ditiotreitol

ELISA (“enzyme-linked immunosorbent assay”) – ensaio imunoenzimático

emPAI (“exponentially modified Protein Abundance Index”) - índice de abundância de proteína

modificado exponencialmente

ESCRT (“endosomal sorting complex required for transport”) – complexo de endereçamento

endossomal necessário ao transporte

ESI-MS (“electronspray ionization mass spectrometry”) – ionização por bombardeamento de

electron acoplado a espectometria de massas

FCS (“fetal cow serum”) – soro fetal bovino

FGF2 (“fibroblast growth factor 2”) – fator de crescimento de fibroblasto 2

FITC (“fluorescein isothiocyanate”) – isotiocianato de fluoresceína

GlcCer - glucosilceramida

GPI - glicofosfatidilinositol

GRASP (“General receptor for phosphoinositides 1-associated scaffold protein”) – proteína de

arcabouço associada a receptor de fosfoinositídeos 1

GXM - glucuronoxilomanana

HIV (“human immunodeficiency virus”) – vírus da immunodeficiência humana

HPTLC (“high performance thin layer chromatography”) – cromatografia em camada fina da alta

performance

Hsp (“heat shock protein”) – proteína de choque térmico

IEM –(“immunoelectronmicroscopy”) – microscopia imunoeletrônica

IFN-γ – interferon - gamma

IL – interleucina

ILVs (“intraluminal vesicles”) – vesículas intraluminais

LDH – lactato desidrogenase

L-NIL - N6-(1-Iminoetil)-L-lisina dihidrocloreto

Mab 18B7 – anticorpo monoclonal 18B7

MIF – (“macrophage migration inhibitory factor”) - fator inibidor de migração de macrófagos

NO (“nitric oxide”) – óxido nítrico

OMVs (“outer membrane vesicles”) – vesículas de membrane externa

PC (“phosphatidylcholine”)- fosfatidilcolina

PRR (“pattern recognition receptors”) – receptores de reconhecimento de padrões

PVP - polivinilpirrolidona

RE – retículo endoplasmático

rf – índice de retenção

TEM (“transmission electron microscopy”) – microscopia eletrônica de transmissão

TGF- β (“transforming growth factor beta”) – fator de transformação de crescimento beta

TGN (“trans Golgi network”) – rede trans Golgi

TNF-α (“tumor necrosis factor alpha”) – fator de necrose tumoral alpha

ts (“temperature- sensitive”) - sensível a temperatura

t- SNARE (“trans SNAP (Soluble NSF Attachment Protein) Rceptor”) – receptor para SNAP trans

UFC – unidade formadora de colônia

Vps (“vacuolar sorting protein”) – proteína de endereçamento para vacúolo

v- SNARE (“vesicle SNAP (Soluble NSF Attachment Protein) Rceptor”) – receptor para SNAP

vesicular

WT (“wild type”) – tipo selvagem

xii

ÍNDICE

I-Introdução……………………………………………………………………………………………………………………………….1

1. Mecanismos clássicos de secreção: aspectos históricos………………………………………………….1

2. A parede celular fúngica…………………………………………………………………………………………………7

3. Mecanismos não-convencionais de secreção…………………………………………………………………8

4. Cryptococcus neoformans: patógeno e modelo de estudo……………………………………………13

5. Resposta immune anti-C. neoformans………………………………………………………………………….22

II- Objetivos…………………………………………………………………………………………………………………………...27

III- Materiais e Métodos…………………………………………………………………………………………………………..29

IV- Resultados………………………………………………………………………………………………………………………….47

Anexo 1………………………………………………………………………………………………………………………..56

Anexo 2……………………………………………………………………………………………………………………..…69

Anexo 3……………………………………………………………………………………………………………………..…78

Anexo 4………………………………………………………………………………………………………………………..88

V- Discussão…………………………………………………………………………………………………………………………..117

VI- Referências Bibliográficas…………………………………………………………………………………………………126

VII- Anexo 5…………………………………………………………………………………………………………………………...148

I – Introdução:

1. Mecanismos clássicos de secreção: aspectos históricos

O trabalho pioneiro que descreveu o transporte e secreção de proteínas em células

eucariotas foi desenvolvido por George Palade e conferiu a ele o prêmio Nobel de Fisiologia em

1974. Havia sido descoberto através de estudos morfológicos e de fracionamento celular que as

proteínas atravessam uma complexa rede de membranas intracelulares até serem secretadas e

que vesículas de transporte estabelecem a comunicação entre estas membranas (Palade, 1975).

Este trabalho levantou diversas questões sobre como essas vesículas eram criadas, como

atingiam o compartimento correto e como os diferentes compartimentos mantinham sua

identidade a despeito da constante fusão de vesículas de transporte (Schekman, 2002). Coube a

Randy Sheckman e colaboradores o desafio de elucidar os mecanismos envolvidos em transporte

de vesículas e secreção. Para este estudo, foi escolhida a levedura Saccharomyces cerevisiae

como organismo modelo devido a facilidade de manipulação genética. A partir de uma triagem

em mutantes sensíveis a temperatura (ts, do termo em inglês “temperature-sensitive”) foi

possível identificar 23 genes envolvidos na secreção de proteínas (Novick, et al. 1980). Esta

técnica é utilizada no estudo de genes essenciais pois o fenótipo mutante é expresso somente a

C, uma vez que a proteína apresenta mutações pontuais que causam a perda de estrutura e

portanto de função a 37

C. Tais mutantes apresentavam acúmulo de vesículas citoplasmáticas

na temperatura de restrição (Novick, et al. 1980). Mais do que isso, diferentes mutantes

apresentavam diferentes aspectos morfológicos, pois presumivelmente acumulavam vesículas

em diferentes pontos da via de secreção (Figura 1).

Figura 1. Micrografias eletrônicas de mutantes sec de S. cerevisiae em temperatura restritiva. Os

mutantes apresentam acúmulo de vesículas de transporte (A), extensão de RE (B) e expansão do

Complexo de Golgi (Bb – “Barkeley Body”). Símbolos: ves, vesículas; va, vacúolo; er, retículo

endoplasmático; n, núcleo; nm, membrana nuclear; np, poro nuclear. Imagem reproduzida de Novick, et

al. 1980.

Proteínas destinadas à membrana plasmática ou ao ambiente extracelular engajam a

chamada via biossintética secretória (Bonifacino & Glick, 2004). Aquelas endereçadas ao vacúolo

e endossomos compartilham os estágios iniciais desta via (Figura 2). Tais proteínas apresentam

uma seqüência sinal para síntese no retículo endoplasmático (RE) na sua extremidade N-

terminal. Após a tradução acoplada ao transporte para o lúmen do RE, as proteínas são

transportadas ao Complexo de Golgi por carreadores vesiculares (Figura 2) (Bonifacino &

Lippincott-Schwartz, 2003). Após receberem modificações pós-transcricionais são endereçadas

ao destino final (por exemplo, membrana plasmática ou endossomos) a partir da rede trans-Golgi

(TGN) (Figura 2).

O brotamento de vesículas do compartimento doador bem como a incorporação seletiva de

proteínas cargo requer o recrutamento de “proteínas de revestimento” (coat proteins) as quais

compõem complexos supramoleculares capazes de deformar a membrana do compartimento e

induzir o brotamento (Figura 3) (Kirchhausen, 2000). Os principais complexos de revestimento

que operam na via secretória são COPII, o qual reveste vesículas que brotam do RE e se fundem

ao Cis-Golgi (Waters, et al. 1991; Barlowe, et al. 1994), COPI, responsável pelo transporte intra-

Golgi e pelo transporte retrogrado Golgi-ER e Clatrina, que reveste vesículas que brotam da rede

trans-Golgi (TGN) e da membrana plasmática (Figura 2) (Letourneur, 1994). Depois de formada, a

vesícula libera o complexo de revestimento que estará disponível no citosol para formação de

uma nova vesícula (Figura 3, passo 4). A perda do complexo se dá pela ação de enzimas

acessórias (Bonifacino & Glick, 2004).

Figura 2. Vias de tráfego intracelular e os principais compartimentos envolvidos. As setas indicam os

possíveis caminhos percorridos por vesículas de transporte. As cores indicam a localização dos complexos

de revestimento COPII (azul), COPI (vermelho) e clatrina (amarelo) (Adaptado de: Bonifacino & Glick,

2004). A via biossintética- secretora carreia moléculas sintetizadas no RE e modificadas no Golgi para o

ambiente extracelular ou para demais compartimentos intracelulares. A via endocítica carreia moléculas a

partir da superfície celular para os lisossomos/vacúolo e vesículas que brotam do Golgi também podem

engajar a via endocítica.

Diversas classes de proteínas funcionam no contato inicial entre as membranas da vesícula e

do compartimento aceptor e são coletivamente chamadas de proteínas de ancoramento

(“tethering proteins”). O principal tipo de reconhecimento se dá através de proteínas

transmembrana contendo um longo domínio super-hélice que reconhecem Rab-GTPases

específicas (conhecidas como proteínas Ypt em leveduras) ligadas à membrana das vesículas

(Figura 3, passo 5) (Segev, 2001; Jahn, et al. 2003).

Figura 3. Mecanismos de brotamento e fusão de vesículas de transporte. As proteínas de revestimento

formam um complexo na membrana do compartimento doador (1). O complexo de revestimento induz

brotamento na membrana e a vesícula se forma contendo proteínas cargo solúveis e transmembrana vem

como a v-SNARE específica deste compartimento (2) A cisão da vesícula ocorre com auxílio de proteínas

acessórias (3). A vesícula perde o revestimento em conseqüência da inativação de GTPases existentes no

complexo de revestimento e da ação de outras enzimas (4). A vesícula é guiada ao compartimento

aceptor, provavelmente através de componentes do citoesqueleto. O reconhecimento do compartimento

aceptor ocorre pela ligação de uma Rab-GTPase específica da vesícula a um “fator de reconhecimento”

presente na membrana alvo (5). A ligação de v-SNARE a t-SNARE também é fundamental para o

reconhecimento (6) além de mediar a fusão das membranas (7). Após a fusão as proteínas SNAREs se

dissociam e v-SNARE é reciclada para o compartimento doador através do mesmo mecanismo (Adaptado

de Bonifacino & Glick, 2004).

A fusão de membranas requer mecanismos redundantes de reconhecimento para garantir a

alta fidelidade do processo (Bonifacino & Glick, 2004). Por isso, proteínas SNAREs também

operam no reconhecimento do compartimento receptor (Figura 3, passos 6 e 7). Foi proposto

que vesículas destinadas a um dado compartimento carreiam em sua membrana uma v-SNARE

específica, a qual reconhece uma t-SNARE cognata na membrana do compartimento receptor

(Rothman, 1994). Estas proteínas são ancoradas as membranas pela porção C-terminal, possuem

“motivos SNAREs” repetidos de 60-70 aminoácidos que auxiliam a formação de uma super-hélice

e apresentam o N-terminal voltado para o citosol (Bock, et al. 2001). Estudos estruturais

mostraram que a ligação entre v- e t-SNAREs forma um feixe de 4 hélices bastante estável

(Sutton, et al. 1998), sendo um monômero de v-SNARE e um trímero de t-SNARE. Além do

reconhecimento, SNAREs medeiam fusão de membranas, processo energeticamente

desfavorável pois requer a remoção de moléculas de água entre as duas superfícies hidrofílicas

das membranas. A formação do feixe de super-hélices parece suprir a energia livre necessária

para fundir duas bicamadas lipídicas (Figura 4) (Weber, et a. 1998). Uma vez que houve a fusão, o

complexo cis-SNARE (v- e t- SNAREs associadas na mesma membrana) deve se dissociar, processo

que requer o recrutamento de proteínas NSamF e α-SNAP e hidrólise de ATP (Sutton, et al. 1998;

Rice & Brunger, 1999). v-SNAREs são então empacotadas em novas vesículas e recicladas ao

compartimento anterior (Bonifacino & Glick, 2004).

Figura 4. A. Mecanismo de fusão de vesículas de transporte

mediado por proteínas SNAREs. Um forte pareamento entre

SNAREs força a íntima aposição das duas bicamadas lipídicas de

modo que as moléculas de água sejam expelidas da interface. B.

Estrutura cristal das duas super-hélices complexadas (v- e t-

SNAREs) (adaptado de Sutton, et al. 1998).

Como se pode perceber, o funcionamento correto da via biossintética-secretória depende de

uma extensa maquinaria molecular que deve garantir (i) que as moléculas-cargo sejam

selecionadas das residentes no compartimento doador, (ii) que as vesículas de transporte se

formem, (iii) que as vesículas cheguem ao compartimento aceptor e (vi) que a maquinária de

transporte seja reciclada ao compartimento doador (Stephens & Pepperkok, 2001). A via

endocítica segue as mesmas regras utilizando uma maquinária molecular totalmente distinta. A

endocitose capta macromoléculas no fluído extracelular a partir da formação de vesículas

revestidas por Clatrina na membrana plasmática (Figura 2). Estas podem ser encaminhadas aos

endossomos de reciclagem e retornam a membrana plasmática. Alternativamente, as vesículas

são encaminhadas ao endossomo inicial, o qual sofre maturação e acidificação do conteúdo

enquanto recebe hidrolases e H

-ATPaes lisossomais advindas do Complexo de Golgi. Neste

estágio, o compartimento endocítico é chamado endossomo tardio e funde-se ao lisossomo/

vacúolo para completar a degradação do conteúdo endocitado (Figura 2) (Kornfeld & Mellman,

1989).

Em células fúngicas a via secretória confronta-se com uma barreira adicional: a parede

celular. Células vegetais enfrentam o mesmo problema, entretanto, sabe-se que uma estrutura

oriunda da membrana plasmática atravessa a parede celular permitindo a troca de

macromoléculas entre células vizinhas. Tal estrutura denomina-se plasmodesmo (Hyun & Uddin,

2010). Em procariotos os mecanismos de secreção através da parede são amplamente

conhecidos e envolvem múltiplos genes (SECA, SECY, SECE E SECG) e diversas proteínas

acessórias (Driessen, et al. 1998). Em bactérias gram negativas, seis diferentes mecanismos de

secreção foram identificados (sistemas de secreção do tipo I ao VI). Estes sistemas operam

liberando proteínas no espaço extracelular e no caso de patógenos, injetando toxinas na célula

hospedeira (Bingle et al. 2008; Cianciotto, 2005; Craig & Li, 2008). Em eucariotos, embora a

maioria dos estudos de mecanismos de trágefo tenham se desenvolvido em leveduras,

pouquíssimo é conhecido sobre o transporte de macromoléculas através a rígida estrutura da

parede celular de fungos.

2. A parede celular fúngica:

A parede celular é um envoltório de superfície que confere rigidez, forma e proteção contra

estresse físico e lise osmótica, além de contribuir com a regulação da permeabilidade celular

(Kapteyn, et al. 1999). A parede celular fúngica é formada basicamente por β1-3 e β1-6 glucanas,

além de polímeros de β1-4-N-acetilglucosamina denominados quitina e proteínas altamente

glicosiladas conhecidas como CWP (Cell Wall Proteins) (Klis, et al 2002). A maioria das CWPs é

transportada para superfície em ligação à âncora de glicofosfatidilinositol (GPI), posteriormente

sofrem clivagem e incorporação à parede celular através de ligação a uma porção remanescente

da âncora (Muller, et al. 1997). Embora a estrutura da parede celular seja rígida, é fato que essa

estrutura é suficientemente plástica para permitir o crescimento celular induzido pela pressão de

turgor (Levin, 2005). Para isso, a parede celular sofre constante remodelamento, o qual é

acompanhado pelo endereçamento de vesículas secretórias contendo componentes da parede

celular e enzimas como glucanases e quitinases (Novick & Schekman, 1979, 1983; Levin, 2005).

Este remodelamento é um processo finamente regulado e depende da polimerização de actina

(Drubin & Nelson, 1996). Alguns fungos podem apresentar ainda melanina na parede celular, a

qual confere maior proteção contra os estresses ambientais (Gomes & Nosanchuk, 2003). Esse

pigmento é também determinante para a porosidade da parede (Jacobson & Ikeda, 2005). A

melanina é sintetizada a partir de compostos fenólicos (Gomez & Nosanchuk, 2003) por

mecanismos descritos nas próximas seções.

Moléculas aparentemente atípicas são freqüentemente encontradas na parede celular de

fungos, como proteínas de choque térmico (hsp), glicoesfingolipídeos e histonas (Chaffin, et al.

1998; Rodrigues, et al. 2000; Nosanchuck et al. 2003; Nimrichter, et al. 2005). Tal diversidade

molecular sugere que existam mecanismos não-convencionais de endereçamento destas

moléculas para fora da célula.

3. Mecanismos não-convencionais de secreção:

A secreção de proteínas a partir do eixo RE-Golgi é extremamente acurada e eficiente e

carreia a maioria das proteínas destinadas ao espaço extracelular. Estas apresentam o peptídeo

sinal que dirige a tradução acoplada ao transporte para o interior do RE, conforme mencionado

anteriormente. Entretanto, diversas proteínas sabidamente encontradas no ambiente

extracelular não apresentam o peptídeo sinal, sendo sintetizadas em ribossomos livres no citosol.

Exemplos dessas proteínas são: fator-α de leveduras, FGF2 (fibroblast growth factor 2), MIF

(macrophage migration inhibitory factor), AcbA (acyl-coenzyme A binding protein) e IL-1β

(revisado em Nickel & Rabouille, 2009). Os processos relacionados ao transporte de tais

proteínas para o ambiente extracelular são coletivamente conhecidos como secreção não-

convencional de proteínas. Neste sentido, diferentes mecanismos estão implicados. O fator-α,

por exemplo, utiliza um transportador transmembrana da família ABC (ATP-binding cassette)

(McGrath & Varshavsky, 1989). A citocina IL1- β é abundantemente secretada durante a resposta

inflamatória, uma vez que caspase 1 é ativada e cliva a forma precursora de IL1- β gerando a

proteína madura (Andrei, et al. 1999). Tem sido proposto que IL1-β é translocada para o interior

de lisossomos secretórios e carreada para o espaço extracelular após a secreção lisossomal

(Andrei, et al. 1999; Andrei, et al. 2004). Todavia, mecanismos de secreção dependentes de

microvesículas também devem estar implicados. Foi descrito que vesículas extracelulares

denominadas exossomos contém IL1-β na forma ativa (Qu, et al. 2007).

Exossomos são vesículas liberadas no espaço extracelular que apresentam entre 40-100 nm e

possuem origem endossomal (Figura 5). A composição lipídica dos exossomos é uma de suas

características marcantes, sendo estes enriquecidos em colesterol, esfingolipídios e

glicerofosfolipídios contendo ácidos graxos de cadeia longa (Simons & Raposo, 2009). Exossomos

correspondem a vesículas intraluminais (ILVs – intraluminal vesicles) do compartimento

endossomal conhecido como corpo multivesicular (MVB – multivesicular body). MVBs são

compartimentos intermediários da via endocítica que se fundem ao lisossomo/vacúolo para

degradação de seu conteúdo (Simons & Raposo, 2009). Alternativamente, MVBs podem fundir-se

com a membrana plasmática resultando na exocitose das ILVs (Figura 5), então denominadas

exossomos (Schorey & Bhatnagar, 2008). A secreção de exossomos é um mecanismo de secreção

não-convencional descrito em diversos tipos celulares como reticulócitos (Pan, et al. 1985),

linfócitos B (Raposo, et al. 1996), células dendríticas (Zitvogel, et al. 1998) e neurônios (Marzesco,

et al. 2005).

A formação das ILVs ocorre a partir de invaginações da membrana limitante do MVB e o

endereçamento para MVB envolve monoubiquitinação da proteína cargo (Figura 6)(Katzmann, et

al. 2004). Entretanto, algumas proteínas que não possuem sítio de ubiquitinação são

endereçadas ao MVB pois são preferencialmente segregadas em microdomínios de membrana

do tipo lipid rafts (de Gassart, et al. 2003). A formação de três complexos hetero-oligoméricos

precede a biogênese do MVB: ESCRT-I, ESCRT-II e ESCRT-III (endosomal sorting complex required

for transport). Estes complexos são formados majoritariamente por proteínas Vps (vacuolar

sorting protein). Mais de 60 produtos gênicos envolvidos no tráfego de proteínas entre Complexo

de Golgi e vacúolo foram identificados em leveduras, sendo o subset Vps classe E responsável

pelo endereçamento ao MVB (Hurley & Emr, 2006). Mutantes vps- classe E exibem acúmulo de

membranas endossomais e defeitos na formação das ILVs (Bowers & Stevens, 2005). Uma vez

que os complexos ESCRT são montados sobre a face citosólica do endossomo, ocorre o

brotamento de vesículas internas (Figura 6). A dissociação da maquinária ESCRT ocorre pela

ação da proteína Vps4 (Yeo, et al. 2003). Estudos de proteômica revelam a presença de diversas

proteínas citosólicas nestas vesículas, tais como actina, cofilina e piruvato quinase, o que é

topologicamente viável, uma vez que invaginações internas podem carrear aleatoriamente o

conteúdo citoplasmático para o interior das ILVs (Schorey & Bhatnagar, 2008).

Diversos estudos têm investigado a função de exossomos em diferentes modelos celulares.

Primeiramente, foi proposto que este mecanismo estaria implicado na eliminação de proteínas

Figura 5. Exossomos são liberados após a fusão

exocítica de MVBs com a membrane plasmática.

O experimento de pulso e caça com BSA

conjugado a partículas de ouro revelou que 20

minutos após a endocitose exossomos contendo

BSA-Gold foram liberados (Simons & Raposo,

2009). Símbolos: PM, membrana plasmática;

MVB, corpos multivesiculares.

obsoletas durante a maturação e diferenciação de reticulócitos (Johnstone, et al. 1984). Em

células dendríticas e linfócitos B, os exossomos participam da apresentação de antígenos

(Raposo, et al. 1996, Zitvogel, et al. 1998). Este mecanismo de apresentação parece ser

especialmente relevante no desenvolvimento da imunidade anti-tumoral (Wolfers, et al. 2001).

Foi demonstrado ainda que exossomos carreiam RNA mensageiros, os quais são transportados

para células vizinhas onde sofrem tradução (Valadi, et al. 2007), estabelecendo um novo modelo

de troca de material genético. Além disso, acredita-se que a liberação de exossomos é um dos

mecanismos de vigilância imunológica de patógenos intracelulares, uma vez que células

infectadas com Mycobacterium sp. (Bhatnagar, et al. 2007b; Beatty, et al. 2000), Salmonella

typhimurium (Bhatnagar, et al. 2007a) ou Toxoplasma gondii (Bhatnagar, et al. 2007a) produzem

exossomos contendo padrões moleculares destes patógenos, os quais são capazes de induzir

uma resposta pro-inflamatória em macrófagos não-infectados.

Recentemente foi descrito que células vegetais são capazes de produzir vesículas

extracelulares semelhantes a exossomos, a despeito da presença de parede celular (Regente et

al. 2009). Análises genômicas comparativas revelaram que alguns gêneros do domínio Archaea

apresentam proteínas homólogas ao complexo ESCRT-III, as quais atuam no remodelamento de

membranas e formação de vesículas (Makarova, et al. 2010). De fato, vesículas extracelulares

similares a exossomos foram isoladas de Sulfolobales e de algumas espécies de Thermococci

durante a resposta a estresse e a infecções virais (Ellen, et al. 2009; Soler, et al. 2008). Bactérias

também desenvolveram mecanismos de secreção vesicular, os quais parecem ser potentes

mecanismos de virulência em bactérias patogênicas (Kuehn & Kesty, 2005). Em bactérias Gram

negativas, a biogênese destas vesículas ocorre a partir da membrana externa à parede celular e

são chamadas OMVs (outer membrane vesicles). Fatores de virulência como β-lactamase,

fosfolipase C e fosfatase alcalina são encontradas nas OMVs de Pseudomonas aeroginosa

(Bomberguer, et al. 2009). Outras bactérias como Helicobacter pylori e Vibrio cholera também

secretam fatores de virulência via OMVs (Yonezawa, et al. 2009; Roy, et al. 2010). Por outro lado,

bactérias Gram positivas como Staphylococcus aureus e Bacillus anthracis também secretam

vesículas extracelulares, entretanto sua via de biogênese não é conhecida (Lee, et al. 2009;

Rivera, et al. 2010).

Figura 6. Papel dos complexos ESCRT na formação de MVBs. O receptor de superfície hipotético é

monoubiquitinado e ocorre formação do endossomo. Diversos componentes dos complexos ESCRT- I, -II e

-III se reúnem na face citosólica do endossomo e são responsáveis por promoverem invaginações de

membrane que levam a formação das ILVs. Complexos ESCRT também são responsáveis por manter a

especificidade das proteínas-alvo destinadas a esta via. (Adaptado de: Alam & Sundquist, 2007).

Outro mecanismo de secreção não-convencional proposto recentemente é dependente da

proteína GRASP (General receptor for phosphoinositides 1-associated scaffold protein) (Kinseth,

et al. 2007). Originalmente foi proposto que a GRASP localiza-se na matriz do Aparato de Golgi,

funcionando na manutenção da organização das cisternas (Barr, et al. 1997; Shorter, et al. 1999).

Entretanto diferentes trabalhos sugerem que GRASP apresenta uma segunda função em

mecanismos de secreção não-convencional (Kinseth, et al. 2007; Duran, et al. 2010, Manjithaya,

et al. 2010). Foi demonstrado que em Dictyostelium discoideum a proteína ligadora de acil-

coenzima A (AcbA) é secretada por meios não convencionais (Kinseth, et al. 2007). Fora da célula,

AcbA é clivada e gera um peptídeo que sinaliza para indução de esporulação (Anjard & Loomins,

2005). Mutantes deficientes na expressão da proteína GrpA (ortólogo de GRASP de mamíferos)

são incapazes de secretar AcbA e de induzir esporulação (Kinseth, et al. 2007). Posteriormente,

esse evento foi avaliado em leveduras e alguns outros componentes desta via não-convencional

de secreção foram identificados, como os genes relacionados à autofagia ATG5, ATG7, ATG8 e

ATG12; a t-SNARE de membrana plasmática SSO1 e os componentes da via de formação de MVB

VPS 23 E VPS4 (componentes dos complexos ESCRT-I e –III, respectivamente) (Duran, et al. 2010).

Corroborando com estes dados, a proteína GRASP também parece ser importante na secreção

do homólogo de AcbA em Pichia pastoris (Manjithaya, et al. 2010). Acredita-se que a secrecão de

AcbA envolva a formação de autofagossomas, os quais convergem com a via de formação de

MVBs sendo então exocitados juntamente com os exossomos. Entretanto o papel da proteína

GRASP neste processo permanece obscuro (Duran, et al. 2010).

4. Cryptococcus neoformans: patógeno e modelo de estudo

C. neoformans é um fungo da ordem Basidiomycota, leveduriforme, agente etiológico da

criptococose. A principal característica morfológica deste fungo é a presença de uma cápsula

polissacarídica que circunda toda a célula a qual pode ser facilmente evidenciada por coloração

negativa usando tinta Nanquim (Figura 7). As cepas de C. neoformans são agrupadas em duas

variedades, de acordo com características sorológicas, bioquímicas e genéticas sendo estas: C.

neoformans var. neoformans (sorotipo D) e C. neoformans var. grubii (sorotipo A). O complexo

Cryptococcus inclui ainda outra espécie patogênica, o C. gattii (dividida nos sorotipos B e C)

(Franzot, et al. 1998, Steenbergen & Casadevall, 2003). C. gattii ocorre em áreas subtropicais e

pode infectar indivíduos imunocompetentes. Já o C. neoformans é um microrganismo

cosmopolita saprófito que causa infecções oportunistas em indivíduos deficientes em imunidade

celular.

A criptococose é contraída pela inalação de esporos ou leveduras dessecadas

(Chayakulkeeree & Perfect, 2006) e se estabelece inicialmente como uma pneumonia

assintomática. Estima-se que 99% da população apresenta anticorpos contra proteínas de C.

neoformans sem apresentar histórico clínico de infecção (Chen, et al. 1999). Acredita-se que

eventualmente a infecção pode permanecer em um estado de latência, e uma vez que o

hospedeiro se torna imunocomprometido a infecção assintomática pode ser reativada, causando

doença (Casadevall & Perfect, 1998). Embora o C. neoformans seja capaz de infectar a maioria

dos hospedeiros imunocompetentes, o desenvolvimento da criptococose ocorre em poucos

Figura 7. Microscopia óptica de campo claro de

uma levedura de C. neoformans contrastada com

tinta Nanquim em magnificação de 100x. O halo

branco em torno do corpo celular é o espaço de

repulsão do nanquim ocupado pela cápsula.

indivíduos, pois depende do estado imunológico do hospedeiro (Steenbergen & Casadevall,

2003). Estima-se que ocorra um milhão de casos de criptococose por ano em indivíduos HIV-

positivos, dos quais 600.000 são fatais (Park, et al. 2009). Em regiões mais carentes de assistência

médica, a sobrevivência média desses pacientes após o diagnostico da criptococose é de menos

de um mês (French, et al. 2002). Outros grupos de pacientes susceptíveis tem surgido como

aqueles submetidos a tratamentos com drogas anti-neoplásicas, submetidos a transplante de

órgãos, e hemodiálise (Casadevall & Perfect, 1998). Uma vez que a infecção pulmonar não é

contida, o microorganismo ganha a corrente sanguínea, se dissemina e atinge o sistema nervoso

central (CNS) causando um quadro grave de meningoencefalite criptocócica, o qual envolve

inflamação no espaço subaracnóide e no parênquima cerebral (Steenbergen & Casadevall 2003).

Para entender de que maneira ocorre esta doença, é importante conhecer as características

do C. neoformans que o tornam um patógeno. Neste sentido, existem fatores de virulência bem

definidos que seguem os requisitos estabelecidos no postulado molecular de Koch (Falkow,

1988). Estas são moléculas produzidas pelo C. neoformans cuja inativação por silenciamento ou

mutação do(s) gene(s) responsável(is) por sua expressão gera atenuação da virulência em

modelo animal. A complementação desses mutantes com as seqüências gênicas originais gera

restauração da virulência (Steenbergen & Casadevall 2003). Dentre estes fatores, destaca-se o

polissacarídeo capsular majoritário conhecido como glucuronoxilomanana (GXM).

A cápsula do C. neoformans é composta em 90% por GXM, molécula que possui peso

molecular variando entre 1.7 e 7 MDa (McFadden, et al. 2006). Esta molécula consiste em uma

cadeia linear de α-(1,3)-manana contendo um resíduo de ácido β-(1,2)-glucurônico a cada tríade

de manose. Resíduos de manose também podem ser 6-O-acetilados e receberem substituições

de β-(1,2) ou β-(1,4)-xilose (Cherniak and Sundstrom, 1994; Cherniak et al., 1988; McFadden et

al., 2006). A razão molar entre xilose: manose: ácido glucurônico varia de acordo com o sorotipo,

sendo 1:3:1; 2:3:1; 3:3:1 e 4:3:1 para os sorotipos D, A, B e C, respectivamente (Cherniak and

Sundstrom, 1994). A Figura 8 apresenta um esquema representativo da estrutura linear da GXM.

Além da GXM, a cápsula é constituída em 8% pelo polissacarídeo denominado galactoxilomanana

(GalXM) (Bose et al., 2003; Vaishnav et al., 1998), o qual apresenta uma cadeia linear de α-(1,6)-

galactana contendo cadeias laterais oligossacarídicas curtas de α-(1,3)- e α-(1,4)-manana, além

de grupos laterais de β-(1,2)- e β-(1,3)- xilose (Bose et al., 2003; McFadden et al., 2006; Vaishnav

et al., 1998). Especula-se que manoproteínas compõem minoritariamente a cápsula do C.

neoformans. Todavia, sua função na arquitetura capsular é pouco conhecida (Zaragosa, et al.

2009).

A maioria dos polissacarídeos microbianos que compõem a parede celular (tais como quitina

e glucanas) são sintetizados por enzimas presentes na membrana plasmática e durante a

biossíntese os polímeros são diretamente transportados para o espaço periplasmático (Leal-

Morales et al., 1994;

Valdivia & Schekman, 2003; Ortiz &

Novick, 2006). Ao contrário, a GXM é

sintetizada no ambiente intracelular (Feldmesser, et al. 2001) e transportada através de

mecanismos pouco conhecidos. Acredita-se que o transporte de GXM envolva o produto do gene

Figura 8. Um dos possíveis

motivos repetitivos de GXM. As

posições das substituições, bem

como o número de substituições,

variam de acordo com o

sorotipo.

CAP59, entretanto sua função não foi esclarecida (Garcia-Rivera, et al. 2004). A mutação do gene

SAV1, o qual codifica uma Rab-GTPase fundamental para o transporte a partir de TGN, levou ao

acúmulo de vesículas citoplasmáticas contendo GXM em C. neoformans, demonstrando que o

polissacarídeo é sintetizado no Aparato de Golgi (Yoneda & Doering, 2006). Além disso, o

tratamento de C. neoformans com Brefeldina A (um inibidor da via biossintética secretora) levou

a diminuição do diâmetro capsular, confirmando que a síntese de GXM envolve elementos do

Golgi (Hu, et al. 2007). A simples fusão de vesículas de transporte a membrana plasmática

permite que a GXM atinja o espaço periplasmático, entretanto não explica de que maneira o

polissacarídeo de alto peso molecular atravessa a parede celular. Estruturas semelhantes a

vesículas atravessando o espaço periplasmático foram observadas por criofratura e por TEM

(Takeo, et al. 1973; Rodrigues, et al. 2000), o que pode ser um indício de que C. neoformans

apresenta um mecanismo sofisticado de transporte de macromoléculas através da parede

celular.

Diversos estudos mostraram que a cápsula é um importante fator de virulência (Zaragoza, et

al. 2009). Os primeiros genes estão implicados na arquitetura capsular foram CAP59, CAP64,

CAP60 e CAP10. O primeiro a ser identificado foi o CAP59, uma vez que sua deleção por

recombinação homóloga resultou em um fenótipo acapsular e na perda da virulência em modelo

murino (Chang & Kwon-Chung, 1994). Embora sua função não seja totalmente esclarecida, sabe-

se que a proteína codificada pelo CAP59 apresenta homologia com uma α-1,3-manosiltransferase

(Sommer, et al. 2003). Além disso, CAP59 apresenta um provável domínio transmembrana e

estudos de ultraestrutura revelaram papel no transporte intracelular de GXM (Garcia-Rivera et

al., 2004). As deleções de CAP64, CAP60 e CAP10 também resultaram em fenótipo acapsular e

perda de virulência (Chang et al. 1996, Chang & Kwon-Chung, 1998, Chang & Kwon-Chung, 1999),

entretanto pouco se sabe sobre a função destas proteínas. Cap10p possui homologia de

seqüência com uma xilosiltransferase (Klutts et al., 2007) e quando conjugada a GFP apresenta-

se distribuída pelo citoplasma em grupos que assemelham-se a vesículas citoplasmáticas (Chang

& Kwon-Chung, 1999).

A cápsula está envolvida em diversos mecanismos de patogenicidade, incluindo proteção

contra fagocitose, modulação da resposta imunológica, propagação do fungo em tecidos

hospedeiros, e causa de danos de diversa natureza (Zaragoza, et al. 2009). É proposto que a

cápsula polissacarídica mascara estruturas da parede celular que seriam associadas a receptores

de reconhecimento de padrões (PRRs) (Vecchiarelli, 2000). A fagocitose do C. neoformans é

dependente de opsoninas como anticorpos (Kozel, et al. 1998) e iC3b (Kozel & Pfrommer, 1986).

A capacidade de sobreviver no interior de células fagocíticas é um mecanismo central na

patogênese do C. neoformans que parece ter sido adquirido ao longo da evolução através do

contato com predadores ameboides no meio ambiente (Feldmesser, et al. 2001). De fato, o C.

neoformans é considerado um patógeno intracelular facultativo capaz de explorar o ambiente

intracelular para replicação (Feldmesser, et al. 2000) (Figura 10). A cápsula polissacarídica parece

desempenhar um papel chave no parasitismo intracelular, uma vez que mutantes acapsulares

não são capazes de se replicar dentro de células fagocíticas (Feldmesser, et al. 2000). O escape

do C. neoformans durante a interação com fagócitos também está associado a um mecanismo de

extrusão, no qual ocorre replicação no interior de macrófagos e liberação dos fungos para o

espaço extracelular sem que haja danos, tanto para o fungo quanto para a célula hospedeira

(Alvarez & Casadevall, 2006; Ma, et al. 2006). Um mutante acapsular do C. neoformans não sofre

extrusão; o revestimento do mesmo com GXM exógena, entretanto, resulta na recuperação da

capacidade de extrusão da cepa selvagem (Alvarez & Casadevall, 2006). Além disso, foi

observado por video-microscopia que o C. neoformans é capaz de infectar macrófagos

diretamente a partir de células adjacentes infectadas, sem se expor ao espaço extracelular,

através de mecanismo conhecido como propagação célula-célula (Alvarez & Casadevall, 2007;

Ma, 2007). A infecção de macrófagos pelo C. neoformans leva ao acúmulo de vesículas

citoplasmáticas contendo GXM, o que interfere na função das células hospedeiras tornado-as

menos responsivas (Tucker & Casadevall, 2002).

É notável que durante o cultivo in vitro e na infecção pulmonar ocorre o crescimento

capsular (Littman, 1958; Rivera, et al. 1998). Este fenômeno é relevante durante a infecção, uma

vez que se observou em modelo in vivo que cepas incapazes de induzir o aumento diâmetro

capsular promovem uma eficiente resposta inflamatória nos pulmões e a infecção é contida

(Blackstock & Murphy, 1997; Blackstock et al., 1999). Na última década, o mecanismo de

crescimento capsular foi amplamente estudado. Acreditava-se que a cápsula crescia como

conseqüência do acúmulo de GXM na face externa da parede celular (Pierini & Doering, 2001).

Entretanto, foi demonstrado através de marcação covalente da cápsula que o crescimento se dá

de forma distal, isto é, as fibras de polissacarídeo são secretadas para o ambiente extracelular e

então reincorporadas as fibras pré-existentes (Figura 9) (Zaragoza, et al. 2006). Esta re-

incorporação se dá através de ligações iônicas entre as fibras e cátions divalentes presentes no

meio (Nimrichter, et al. 2007). De fato, altas concentrações de GXM são detectadas na forma

livre em meio de cultura (Frases, et al. 2008). In vivo, o polissacarídeo extracelular solúvel se

acumula na corrente sanguínea provocando diminuição da migração de leucócitos para os sítios

de infecção (Vecchiarelli, 2000).

Figura 9. Observação da cápsula do C. neoformans durante incubação em meio de indução de

crescimento capsular. As estruturas capsulares foram coradas inicialmente em verde por reação com o

componente C3 do sistema complemento, seguindo-se coloração com anticorpo monoclonal anti GXM

(vermelho). Este dado sugere que as fibras de GXM mais antigas permanecem junto ao corpo celular e

que a cápsula cresce de forma distal (Zaragoza, et al. 2006).

A síntese de melanina também tem sido associada à virulência no C. neoformans. Mutantes

albinos gerados por mutagênese randômica apresentaram perda de virulência (Williamson,

1997). A melanina é um pigmento sintetizado pela enzima lacase através da polimerização

oxidativa de compostos fenólicos como L-DOPA, dopamina, epinefrina e noreponefrina

(Williamson, 1997). A melanina encontra-se na parede celular, exercendo um papel protetor

contra estresse oxidativo, nitrosativo e contra agentes antimicrobianos (Polacheck, 1991;

Doering, et al. 1999). A lacase foi definida como fator de virulência uma vez que a deleção do

gene levou a perda de virulência e a subseqüente complementação do gene restaurou a

virulência do C. neoformans (Williamson, 1997). Acredita-se que o neurotropismo do C.

neoformans pode ser parcialmente explicado pela capacidade do fungo de utilizar

neurotransmissores tais quais epinefrina e dopamina como substratos para síntese de melanina

(Steenbergen & Casadevall, 2003). Foi descrito que a secreção de lacase é modulada in vivo,

ocorrendo preferencialmente no cérebro e sendo inibida nos pulmões, o que facilitaria a função

deste fator no neurotropismo (Waterman, et al. 2007).

Fosfolipase B1 também foi caracterizada como fator de virulência no C. neoformans (Cox, et

al. 2001). Esta enzima possui atividade de fosfolipase B, lisofosfolipase e transciclase e todas

estas atividades foram abolidas na cepa mutante deletada no gene PLB1 (Cox, et al. 2001).

Estudos in vivo tanto em camundongos quanto em coelhos demonstraram que a cepa plb1



apresenta perda significativa de virulência quando comparada a virulências das cepas selvagem e

reconstituída (Cox, et al. 2001). Além disso, foi demonstrado que isolados clínicos de C.

neoformans apresentam atividade significativamente maior de PLB1 que isolados do meio

ambiente (Ghannoum, 2000). Um modelo de infecção murina revelou que há uma correlação

entre os níveis de atividade de fosfolipase secretada (baixa, intermediária ou alta) e a virulência

das cepas correspondentes (Ghannoum, 2000). O mecanismo de virulência associado a PLB1

secretada deve envolver a hidrólise de fosfolipídios nas membranas das células hospedeiras. Cox

e colaboradores demonstraram que a cepa plb1



apresenta dificuldade de replicação no interior

de monócitos, o que pode estar relacionado à incapacidade de degradar a membrana do

fagossomo (Cox, et al. 2001). Além disso, um dos principais surfactantes do pulmão é um

substrato de PLB1: dipalmitoil-fosfatidilcolina. A degradação do surfactante pode contribuir no

estabelecimento da infecção (Cox, et al. 2001).

Outra importante molécula definida como determinante de virulência do C. neoformans é a

glucosilceramida (GlcCer) (Rittershaus, et al. 2006). Este glicoesfingolipídio é uma molécula de

superfície sendo encontrada majoritariamente na parede celular (Rodrigues, et al. 2000). A

deleção da enzima glucosilceramida sintase I (GCSI) levou a perda de virulência pelo C.

neoformans, a qual foi restaurada com a complementação do gene (Rittershaus, et al. 2006).

Sabe-se que a GlcCer é altamente imunogênica (Rodrigues, et al. 2000) e que anticorpos

monoclonais anti-GlcCer são capazes de proteger camundongos contra doses letais de C.

neoformans (Rodrigues, et al. 2007). A ligação de anticorpos aos sítios de brotamento na parede

celular inibe diretamente a replicação (Rodrigues, et al. 2000). Acredita-se que a GlcCer participa

do controle do ciclo celular em pH fisiológico, porém o mecanismo de patogenicidade ainda não

está totalmente elucidado (Rittershaus, et al. 2006). Através do mesmo princípio de deleção e

complementação genética, descreveu-se que a enzima urease também é um fator de virulência

no C. neoformans (Cox, et al. 2000). Urease catalisa a degradação de uréia a amônia e

carbamato, alcalinizando localmente o ambiente, entretanto sua contribuição para o mecanismo

de patogenicidade não é conhecida (Casadevall & Perfect, 1998).

Todos os fatores de virulência descritos acima têm em comum o fato de serem secretados

para o ambiente extracelular, onde vão agir destruindo componentes do hospedeiro (Cox, et al.

2001), protegendo o C. neoformans de ataques do sistema imune (Williamson, 1999, Zaragoza, et

al. 2008) ou modulando negativamente a imunidade do hospedeiro (Vecchiarelli, 2000).

5. Resposta imune anti- C. neoformans:

Diversos mecanismos tanto da imunidade inata quanto da adaptativa estão relacionados a

proteção contra a criptococose. Além de barreiras físicas como pele e mucosa do trato

respiratório, o sistema complemento e células fagocíticas efetoras exercem papel fundamental

na resposta não- específica a infecção (revisado em Voelz & May, et al. 2010).

O C. neoformans é capaz de ativar tanto a via clássica (mediada por anticorpo) quanto a via

alternativa do Complemento (via hidrólise espontânea de C3 e deposição na superfície

microbiana) (Kozel, et al. 1998 b). Observações de modelos animais revelaram que cobaios

depletados de proteínas do complemento (de C3 a C9) não são aptos a eliminar a infecção por C.

neoformans (Diamond, et al. 1973; Diamond, et al. 1974). Ambas a vias convergem para a

formação do complexo C3- convertase (C4b2a para via clássica e C3bBb para via alternativa) que

leva a hidrólise de C3 em C3a e C3b. C3b age como uma potente opsonina facilitando a

fagocitose via receptores do complemento (CR1, CR3 ou CR4) (Levitz, et al. 1997) e ao mesmo

tempo compõe o complexo C5 convertase que cliva C5 em C5a e C5b. C5a age juntamente com

C3a como um mediador da inflamação e quimioatraente de células fagocíticas efetoras. C5b

inicia a formação do complexo de ataque a membrana (C5b, C6, C7, C8, C9) (Dessauer, et al.

1984), entretanto o C. neoformans é resistente a lise celular mediada por este complexo

(Shapiro, et al. 2002). Camundongos deficientes em C5 são mais suceptíveis a injeção intravenosa

de C. neoformans e morrem três vezes mais rápido de pneumonia aguda do que camundongos

que expressam C5 (Rhodes, 1985; Rhodes, et al. 1980). Cobaios deficientes em C4 apresentaram

o mesmo tempo de sobrevivência dos selvagens após infecção por C. neoformans, indicando que

a via alternativa do complemento tem maior importância na proteção anti- C. neoformans

(Diamond, et al. 1973). Entretanto foi demonstrado que a via clássica é necessária para que haja

uma cinética ótima de opsonização (Diamond, et al. 1974).

A fagocitose do C. neoformans pode ocorrer via receptores do complemento, receptores Fc e

através do reconhecimento direto de componentes de superfície. A GXM é capaz de se ligar aos

receptores TLR4 e CD14 (Shoham, et al. 2001; Barbosa, et al. 2007) promovendo internalização.

Dímeros TLR2/TLR1 e TLR2/TLR6 também reconhecem GXM, entretanto não se sabe se estes

receptores são capazes de mediar a fagocitose (Fonseca, et al. 2010). Receptores de manose de

células dendríticas (DCs – dendritic cells) e receptores CD18 se ligam a manoproteínas da cápsula

(Pietrella, et al. 2005; Dong & Murphy, 1997).

Neutrófilos parecem contribuir fortemente com a resposta anti- C. neoformans. Desafios com

C. neoformans induzem uma rápida migração de células polimorfonucleares, as quais promovem

uma intensa resposta oxidativa local (Miller & Mitchell, 1991). Foi observado que uma intensa

neutropenia em camundongos está associada a um prolongado período de sobrevivência a

infecção pulmonar pelo C. neoformans, sugerindo que possivelmente os neutrófilos exercem

uma função imunoregulatória além de antimicrobiana (Mednick, et al. 2003).

O C. neoformans desenvolveu estratégias únicas para sobreviver no interior de macrófagos,

sendo considerado um patógeno intracelular facultativo (Feldmesser, et al. 2001) (Figura 10).

Diferentemente de outros patógenos como Listeria monocytogenes e Shigella flexneri, o C.

neoformans sobrevive no interior de fagolisossomos e não necessita de mecanismos de escape

para colonização do citoplasma (Levitz, et al. 1999; Southwick & Purich, 1996). Mais do que isso,

o C. neoformans não promove inibição da fusão fagossomo- lisossomo a exemplo da Legionella

pneumophila, (Horwitz, et al. 1983) nem tampouco interfere com a maturação e acidificação do

fagossomo, como se observa durante a infecção por Histoplasma capsulatum e Micobacterium

spp (Strasser, et al. 1999; Sturgill-Koszycki, et al. 1994). Na realidade, o C. neoformans replica-se

no interior de fagolisossomos e elevações no pH deste compartimento levam a redução de sua

taxa de proliferação (Levitz, et al. 1997). Em última análise, a proliferação do C. neoformans

acarreta em lise celular (Del Poeta, 2004; Feldmesser et al. 2000) (Figura 10). Alternativamente,

mecanismos de extrusão discutidos anteriormente nesta tese permitem que a levedura ganhe o

espaço extracelular novamente (Alvarez & Casadevall, 2006; Ma, et al. 2006). O ambiente

intracelular de macrófagos é claramente benéfico para este microorganismo e acredita-se que

ele permaneça durante anos colonizando este ambiente em um estado de latência (Garcia-

Hermoso, et al. 1999; Goldman, et al. 2001). Além disso, é possível que o C. neoformans se utilize

de um mecanismo de “Cavalo de Tróia” para atravessar a barreira hematoencefálica, sendo

carreados por monócitos para o interior do sistema nervosa central (Charlier, et al. 2009).

Figura 10. Representação esquemática de um macrófago infectado pelo C. neoformans. Após a

fagocitose ocorre a fusão fagolisossomal. A membrana do fagolisossomo se rompe e há o acúmulo de

vesículas citoplasmáticas contendo GXM. O C. neoformans se replica no interior do compartimento

fagolisossomal. Após a lise celular, as leveduras são liberadas no meio extracelular (Adaptado de:

Feldmesser, et al. 2001).

Na infecção por C. neoformans, células dendríticas (DCs) funcionam como as principais

células apresentadoras de antígenos, as quais iniciam uma imunidade adaptativa celular

protetora (Bauman, et al. 2000). DCs induzem a maturação de linfócitos T de maneira mais

eficiente de macrófagos alveolares ou peritoniais (Osterholzer, et al. 2009).

Tanto o componente humoral quanto o celular são importantes na resposta immune

adaptativa ao C. neoformans. A eficiência da resposta humoral é dependente da especificidade

do anticorpo (Mukherjee, et al. 1995). Evidencias de modelos murinos mostram ainda que o

efeito protetor de anticorpos relaciona-se a imunidade mediada por célula T. Camundongos

deficientes em células CD4 positivas ou IFN- γ não são protegidos contra infecção através da

administração passiva de IgG1 protetor, enquanto camundongos deficientes em células CD8

positivas, células NK ou C3 são protegidos (Beenhouwer, et al. 2001; Shapiro, et al. 2002; Yuan,

et al. 1997). Portanto, a imunidade adaptativa humoral faz parte da defesa do hospedeiro e está

integrada a demais elementos do sistema imune inato e adaptativo.

A característica comum dos fatores de risco associados ao desenvolvimento da

criptococose (por exemplo infecção por HIV, tratamento com corticosteróides, leucemia) é a

perda da funcionalidade plena da imunidade adaptativa celular (CMI – cell mediated immunity).

A CMI age no controle da infecção através do efeito citotóxico direto ou regulando a função de

fagócitos e células NK (Levitz, et al. 1994). Os efeitos regulatórios da CMI dependem do status

imunológico do indivíduo infectado. Citocinas do tipo Th1 estão associadas a imunidade

protetora enquanto do tipo Th2 estão associadas a suceptibilidade (Hoag, et al. 1997). O

aumento na produção de IFN-γ e de TNF-α associam-se ao controle da infecção (Flesch, et al.

1989; Kawakami, et al. 1995; Milam, et al. 2007), enquanto camundongos deficientes na

produção de IFN-γ apresentam aumento da carga fúngica (Arora, et al. 2005). Foi demonstrado

recentemente que a citocina IL-17 produzida por células Th17 também exerce importante papel

na sobrevivência de camundongos infectados com C. neoformans (Kleinschek, et al. 2006; Muller,

et al. 2007). Por outro lado, citocinas do tipo Th2 como IL-4 e IL-13 associam-se a uma redução

na capacidade de combater o C. neoformans (Blackstock & Murphy, 2004; Kawakami, et al.

1999). A progressão da infecção em indivíduos HIV-positivos correlaciona-se com perda da

resposta Th1 (Altfed, et al. 2000) e com aumento do perfil de citocinas do tipo Th2 em indivíduos

transplantados (Singh, et al. 2008). Portanto, o balanço Th1-Th2-Th17 é essencial para o controle

da infecção.

II – Objetivos:

 Objetivo Geral:

Os mecanismos pelos quais macromoléculas atravessam a parede celular de fungos ainda são

pouco compreendidos. Esta é portanto uma questão central na biologia celular de organismos

dotados de parede celular. Nesse contexto, esta tese visa caracterizar as estruturas celulares

carreadoras de macromoléculas, bem como a maquinária molecular necessária para que o

transporte através da parede celular ocorra. A caracterização de moléculas transportadas por tal

via e a função deste mecanismo de secreção também foram abordados utilizando S. cerevisiae

como organismo modelo de biologia celular e em modelo de infecção celular pela levedura

patogênica C. neoformans.

 Objetivos específicos:

1) Compreender como moléculas tais como GXM e CMH produzidas pelo C. neoformans

atravessam a parede celular e atingem o espaço extracelular. Caracterizar a existência de

ultraestruturas semelhantes a vesículas visualizadas na parede celular e isolar tais

estruturas dos sobrenadantes de cultura de C. neoformans.

2) Através de crioultramicrotomia, caracterizar ultraestruturas intracelulares envolvidas no

transporte de GXM. Avaliar a existência de subpopulações de vesículas que diferem

quanto a composição.

3) Determinar o impacto das vesículas secretadas pelo C. neoformans na interação com

macrófagos murinos, incluindo a produção de citocinas e indução de atividade

microbicida.

4) Avaliar que genes estão potencialmente envolvidos na formação/liberação de vesículas

extracelulares. Isolar, caracterizar morfologica e bioquimicamente vesículas

extracelulares isoladas de diferentes mutantes de vias do tráfego intracelular utilizando S.

cerevisiae como organismo modelo.

III - Materiais e Métodos:

A. Cultivo de leveduras:

As cepas de C. neoformans utilizadas nesse trabalho foram: ATCC 24067 (sorotipo D), B3501

(sorotipo D), H99 (sorotipo A, isolado clínico), HEC3393 (sorotipo A, isolado clínico) e Cap67

(sorotipo D, mutante acapsular). As células foram mantidas meio Sabouraud, composto por 20%

de dextrose e 10% de peptona. Para o isolamento de vesículas as células foram crescidas em1L

de Meio Mínimo, composto por Dextrose (15 mM), MgSO

(10 mM), KH

(29.4 mM), glicina

(13 mM), and tiamina (3 µM). As células foram cultivadas a temperatura ambiente por 72 horas

sob agitação.

As cepas de S. cerevisiae usadas neste estudo foram RSY255, RSY113, SEY6210 e BY4741, as

quais apresentam fenótipo selvagem (WT). Diferentes mutantes de vias de tráfego intracelular

também foram utilizados, os quais estão listados na Tabela I do Artigo IV e serão descritos a

seguir. As cepas RSY782, SF2642-1D, e RSY954 são respectivamente, mutantes temperatura-

restritivos dos genes essenciais sec1-1, sec4-2 e bos1-1/sec32-1. Estes estão relacionados a via

secretória clássica. As cepas EEY6-2 and EEY9 correspondem a mutantes deletados nos genes

vps23 e snf7/vps32, respectivamente, os quais estão relacionados a via de secreção de

exossomos. A cepa Grh1 é defectiva na expressão da proteína homologa a GRASP Grh1p.

Células WT e mutantes foram pré-inoculadas em meio Sabouraud, por 24 horas a temperatura

ambiente e sob agitação. Para o isolamento de vesículas as células foram crescidas em 1L de

meio Sabouraud por 48h a temperatura ambiente e sob agitação. Para os mutantes temperatura-

restritivos, após 48h de crescimento as células foram centrifugadas e transferidas para 1L de

Sabouraud e incubadas por 16h a 37

C. Diferentes tempos de incubação a 37

C (1, 3 e 16h)

foram utilizados no experimento de cinética de secreção do mutante sec4-2. Controles de

viabilidade celular na temperatura de restrição foram feitos através da contagem de unidades

formadoras de colônia (UFC).

B. Isolamento de vesículas:

Após o crescimento celular nas condições descritas acima, as células foram centrifugadas a

5.000 x g a 4

C por 15 minutos. Os sedimentos foram descartados e os sobrenadantes coletados

e novamente centrifugados sucessivamente a 10.000 x g e 15.000 x g a 4

C por 15 minutos,

garantindo a retirada de células e fragmentos celulares. Os mesmos sobrenadantes foram

filtrados em uma membrana de policarbonato com poro correspondente a 0.8µm e

posteriormente concentrados aproximadamente 20 vezes usando um sistema de ultrafiltração

em membrana com cutoff de 100 kDa. Os sobrenadantes concentrados foram então submetidos

a ultracentrifugação a 100.000 x g por 1h a 4

C. Os sedimentos gerados foram lavados 2 vezes

em PBS, sendo seqüencialmente ressuspensos e ultracentrifugados novamente. Para remover

GXM extravesicular as preparações de vesículas de C. neoformans foram passadas em uma

coluna cromatográfica de afinidade, na qual o anticorpo monoclonal anti GXM Mab18B7 foi

mobilizado em partículas de Sepharose. As frações não ligadas a coluna foram coletadas. O

material contido no sedimento obtido por centrifugação a 100.000 x g foi analisado por

diferentes métodos descritos a seguir. Em todas as análises quantitativas de componentes

vesiculares as preparações foram normalizadas pelo número de células através de contagem em

câmara de Neubauer. Nos estudos de interação com macrófagos, vesículas de C. neoformans

foram quantificadas e normalizadas através da dosagem de esterol pelo kit fluorimétrico Amplex

Red (Molecular Probes). Em diferentes ensaios foram detectados entre 6 e 10 µg de esterol a

partir de 2 L de cultura de C. neoformans, sugerindo que 0.5 fg de esterol são recuperados por

célula. Esta aparente baixa eficiência é esperada já que as vesículas supostamente se rompem no

sobrenadante de cultura para liberação de componentes secretados.

C. Análise de Lipídeos Neutros:

Os sedimentos obtidos após a ultracentrifugação a 100000xg foram submetidos a secagem

por centrifugação a vácuo e posteriormente ressuspensos em uma mistura de CHCl

: metanol:

O na proporção 8/4/3 v/v/v. As misturas foram vigorosamente agitadas. Usualmente, duas

fases se formam. A fase superior corresponde a fração aquosa e contém proteínas e açúcares,

além de outros componentes hidrofílicos. A fase inferior corresponde a fração orgânica e contém

os lipídeos neutros. A fase inferior foi levada a secagem em atmosfera de N

e submetida a

partição de Folch, a qual consiste a partição das moléculas extraídas em uma mistura de CHCl

metanol: KCl 0.25% na proporção 8:4:3. A fase inferior foi então submetida a análise por

cromatografia em camada fina de alta performance (HPTLC). Para análise de esteróis, os estratos

lipídicos foram aplicados em placas de sílica e separados usando um sistema de solventes

contendo hexano: éter: ácido acético na proporção 40: 20: 1 v/v/v. As placas foram borrifadas

com uma solução contendo 0.05% de FeCl

, 5% de ácido acético e 5% de ácido sulfúrico. As

placas foram aquecidas a 100 C por 5 minutos e as bandas equivalentes a esteróis surgiram em

cor violeta. Como controle foi usado um padrão de ergosterol (Sigma) e as bandas que migraram

como mesmo índice de retenção (rf) que o padrão foram identificadas. A presença de

glucosilceramida (CMH) foi avaliada usando o sistema de solventes CHCl

: metanol: H

O na

proporção 65: 25: 4 v/v/v. As placas foram reveladas borrifando uma solução de orcinol sulfúrico

seguido de incubação a 100 C por 3 minutos.

A fase inferior da partição de Folch também foi analisada por espectrometria de massas em

modo de ionização por eletrospray (ESI-MS), utilizando o instrumento Finnigan LCQ DUO ion trap

(Thermo Electron, San Jose, CA). As amostras foram diluídas em CHCl

:metanol 1:1, v/v,

contendo 10mM de iodeto de lítio e injetadas no equipamento de ESI-MS operando em fluxo de

10µl/minuto. A análise foi realizada em modo positivo. As voltagens da fonte e do capilar foram

respectivamente de 4.5 kV e 3V. A temperatura do capilar foi mantida a 200

C. Os espectros

foram coletados em um intervalo de massa sobre carga (m/z) correspondendes a 300 a 800. A

energia de dissociação na fonte utilizada foi de 25 V. Os experimentos de ativação colisional

induzida (MS/MS) foram realizados com percentual de energia entre 20 - 60% (1-3 eV). Todos os

espectros foram processados usando Xcalibur software (Thermo Electron). Os experimentos aqui

descritos foram realizados em equipamento disponível no Instituto de Ciências Biológicas da

Universidade de São Paulo.

D. Análise de Fosfolipídios:

A análise de fosfolipídios foi feita a partir de vesículas isoladas de C. neoformans. O

sedimento de contendo vesículas foi preparado como descrito acima e os lipídeos resultantes

foram ressupensos em 90% de metanol. Alíquotas de 100 µl foram injetadas em cromatógrafo

líquido equipado com uma coluna cromatográfica de fase reversa (Supleco Ascentis Express, C18,

150x2.1mm), equilibrada em solução de metanol: H

O, 95:5, v/v, contendo 1mM de acetato de

amônio. As amostras foram eluídas em concentrações crescentes de misturas de clorofórmio-

água, 500/0,2, v/v, contendo acetato de amônio 1 mM. As frações eluídas foram coletadas

automaticamente (intervalos de 1 minuto) e submetidas a análise por ESI-MS conectada a um

espectrômetro de massas do tipo quadrupolo/tempo de vôo (QTOF-MS, PE Sciex API III

operando no modo de varredura do pico parentalcom m/z de 184,1 (característico de

fosfatidilcolina). Dessa forma, a câmara de colisão foi preenchida com argônio e o detector foi

programado setado para transmitir os sinais oriundos do pico 184.1 m/z. Para confirmar as

assinalações feitas a partir dos dados de LC/MS/MS, e para melhor definir as cadeias alifáticas da

fosfatidilcolina na amostra, espectros de íons positivos e negativos foram coletados a partir da

injeção direta de frações coletadas durante LC/MS/MS. As condições experimentais foram

otimizadas utilizando padrões de di-palmitoil-fosfatidilcolina, heptadecanoil-liso-fosfatidilcolina e

di-heptanoilfosfatidilcolina. Estes experimentos foram feitos utilizando métodos originalmente

descritos por Jensen, et al. (Jensen, et al. 1986) e, nessa tese, foram realizados com a

colaboração do Dr. Kym Faull (Pasarow Mass Spectrometry Laboratory, Semel Institute for

Neuroscience and Human Behavior and the Department of Psychiatry and Biobehavioral

Sciences, David Geffen School of Medicine, University of California, Los Angeles, EUA).

E. Detecção de GXM por ELISA:

Para inferir se as vesículas isoladas do sobrenadante de cultura do C. neoformans continham

GXM, foi usado ELISA de captura. As amostras de vesículas correspondem a fração aquosa após a

extração lipídica. Placas de 96 poços foram revestidas com 1 µg/ml do anticorpo monoclonal

Mab 12A1, uma IgM específica para GXM. Após 1h de incubação a placa foi bloqueada com PBS

1% BSA incubando por 1h a temperatura ambiente. As placas foram lavadas 3 vezes com PBS-

0.05% Tween. GXM purificada foi usada como curva-padrão. Curva-padrão e amostras foram

incubadas por 1h a temperatura ambiente e novamente lavadas 3 vezes com PBS-0.05% Tween.

As placas foram então incubadas com 1 µg/ml do anticorpo de detecção, Mab18B7, uma IgG de

camundongo anti-GXM. Após uma hora de incubação a 25 C as placas foram novamente lavadas

3 vezes e incubadas com 1 µg/ml do anticorpo secundário anti-IgG de camundongo acoplado a

fosfatase alcalina por 1h. Após nova etapa de lavagem as placas foram incubadas com o Tampão

Glicina-pH 9.0 contendo 0,2%de cloreto de zinco e cloreto de magnésio, bem como 0.1% do

substrato p-nitrofenil-fosfato dissódico hexahidratado. Os resultados foram lidos a 405 nm

utilizando um espectrofotômetro.

F. Análise da detecção de vesículas em suspensões de células não viáveis:

Para averiguar se a secreção de vesículas é um fenômeno fisiológico e portanto dependente

da viabilidade celular ou se seria fruto de artefatos oriundos de células mortas, a presença de

lipídeos marcadores de vesículas nos sedimentos 100.000 x g foi avaliada nas seguintes

condições: i) Cultura de C. neoformans foi crescida em Meio Mínimo por 72h; ii) Após o

crescimento por 72h células foram tratadas com 100mM de azida de sódio por 1h a 25 C e iii)

após 72h de crescimento células foram incubadas a 50

C por 1h. As preparações de vesículas

foram normalizadas pelo número de células nas três condições, através de contagem em Câmara

de Neubauer. A viabilidade celular foi acompanhada inoculando-se células controle, tratadas

com NaN

e incubadas a 50

C em placas de Ágar Sabouraud. Após as incubações, as células

foram removidas e as vesículas foram isoladas dos sobrenadantes, conforme descrito acima. Os

sedimentos de vesículas foram submetidos a análise de lipídeos neutros descrita previamente.

G. Ensaio de formação extracelular de vesículas:

A fim de avaliar se as vesículas se formariam de forma randômica como resultado de

agregação aleatória de lipídeos e GXM liberados pelas células, o mutante acapsular do C.

neoformans, Cap67, foi crescido em Meio Mínimo. A este meio, foi adicionada GXM a 15 µg/ml,

concentração previamente estabelecida em sobrenadantes da cepa capsulada. Após 72h de

crescimento sob esta condição, vesículas foram isoladas do sobrenadante. Em paralelo células

da cepa Cap67 foram crescidas na ausência de GXM, a qual foi adicionada na concentração de 15

µg/ml após a remoção das células. Os sobrenadantes contendo GXM foram ultracentrifugados e

o sedimento 100000 x g foi lavado três vezes e submetido a cromatografia de afinidade em

resina de sefarose acoplada ao Mab18B7. A GXM presente nas vesículas, bem como nos

sobrenadantes foi então quantificada por ELISA de captura. Sobrenadante de Cap67 foi usado

como controle negativo.

H. Ensaio de reincorporação de GXM:

A cepa acapsular de C. neoformans Cap67 foi crescida por 48h em Meio Mínimo, lavada 3

vezes em PBS e ajustada para 10

células. Estas, foram incubadas em 100µl de suspensão

vesicular purificada da cepa capsulada HEC3393. Em paralelo, a concentração de GXM desta

mesma amostra foi dosada por ELISA e correspondeu a 10 µg/ml. A suspensão de células e

vesículas foi incubada por 12h a 25

C e então as células foram lavadas extensivamente em PBS e

fixadas em PBS 4% paraformaldeído. As células foram lavadas 3 vezes em PBS, bloqueadas por 1h

em PBS 5% BSA e posteriormente incubadas com o anticorpo anti-GXM MAb18B7 a 1µg/ml por

1h. Após 3 lavagens em PBS, as células foram incubadas com o anticorpo secundário anti- IgG

conjugado a FITC. Laminas foram montadas e as leveduras foram observadas em um microscópio

de fluorescência Axioplan 2 (Zeiss, Alemanha). As imagens foram adquiridas usando uma câmera

digital Color View SX e processadas usando o software analySIS (Soft Image System). Como

controle, o Mab18B7 foi substituído por uma IgG irrelevante. Como controle positivo as células

foram incubadas com GXM solúvel, purificada como descrito previamente (Nimrichter, et al.

2007).

I. Aferição de tamanho de cápsula:

Para induzir a expressão de cápsula no C. neoformans, células foram incubadas em meio

Sabouraud dez vezes diluído em 50mM de MOPS pH 7.3, conforme descrito anteriormente

(Zaragoza & Casadevall, 2004). Após incubação por 6 e 24h a 37

C sob agitação, as células foram

centrifugadas a 2000 x g por 10 minutos e fixadas com PBS 4% de paraformaldeído. Após a

fixação as células foram lavadas com PBS e a cápsula foi evidenciada com tinta nanquim. O

tamanho da cápsula foi medido por microscopia óptica de campo claro. O tamanho da cápsula é

definido como a distância entre a parede celular e a borda externa da cápsula. Esta medida

morfométrica foi determinada usando o software ImageJava (http://rsb.info.nih.gov/ij/). Todos

as medidas foram feitos em triplicatas e foram estatisticamente validados pelo teste t Student’s.

J. Microscopia Eletrônica de Transmissão (TEM):

TEM foi usada para visualizar vesículas tanto de C. neoformans quanto de S. cerevisiae. Foram

analisadas preparações de vesículas isoladas e vesículas associadas a leveduras durante a

infecção de tecido pulmonar in vivo. Para as preparações a partir de vesículas isoladas, os

sedimentos obtidos após as lavagens a 100000xg foram fixadas em 2% de glutaraldeído em 0.1M

de cacodilato a temperatura ambiente por 2h e em seguida foram incubados por 16h em 4% de

formaldeído, 1% de glutaraldeído em PBS. Posteriormente as amostras foram incubadas por 90

minutos em 2% de OsO

, desidratadas serialmente em etanol e embebidas em Spurr. Cortes

ultrafinos foram obtidos em um Reichert Ultracut e contrastados em acetato de uranila 0.5%.

Amostras foram observadas em um microscópio eletrônico de transmissão JEOL 1200EX

operando à 80kV.

Para detecção de vesículas in vivo, foram feitas preparações de tecido pulmonar de

camundongos infectados com C. neoformans da cepa ATCC 24067. Camundongos C57BL/6 foram

infectados com 1x10

leveduras de forma intratraqueal e em seguida sacrificados com 2h, 48h ou

7 dias de infecção. O processamento para TEM foi feito conforme previamente descrito

(Feldmesser, et al. 2001).

K. Crioultramicrotomia e imuno detecção:

Este procedimento foi baseado em estudos pioneiros da década de 70 (Tokuyasu, 1973).

Células foram fixadas em paraformaldeído 4%, glutaraldeído 0.2% e ácido pícrico 1% diluídos em

0.1M de cacodilato de sódio pH 7.2. Após 60 minutos a temperatura ambiente as células foram

lavadas 3 vezes, infiltradas em gelatina, cortadas em cubos de 1mm e infiltradas em

polivinilpirrolidona 25% (PVP) e sacarose 2.3M por 16h a 4

C. Os blocos embebidos em PVP

foram montados em um suporte e congelados rapidamente por imersão em N

líquido.

Criossecções foram obtidas a -130

C em um crioultramicrotomo Ultracut UCT (Reichert). Os

cortes foram coletados com uma alça e transferida para grades de níquel revestidas com

películas de Formivar. Para a imunomarcação, as grades foram bloqueadas com PBS 1% BSA por

1h. As grades foram então incubadas com o anticorpo primário. O anti-GXM de camundongo

Mab18B7 foi usado a 1µg/ml, enquanto anticorpos anti-CMH foram isolados de soro de

pacientes como previamente descrito (Rodrigues, et al. 2000). As grades foram lavadas em PBS

por 3 vezes de 10 minutos e posteriormente incubadas com anti- IgG de camundongo ou anti-

Humano conjugados a partículas de ouro de 15 nm. Após uma série de lavagens em PBS e H

destilada as grades foram deixadas por 15 minutos em 2% de acetate de uranila aquosa e

transferida para uma gota de 0.75% de metilcelulose. Após a secagem das grades, as amostras

foram observadas em um microscópio eletrônico de transmissão JEOL 1200EX operando a 80kV.

L. Fracionamento de vesículas em gradiente de densidade:

Vesículas de C. neoformans foram obtidas por ultracentrifugação como descrito

anteriormente. Para identificar diferentes subpopulações de vesículas um gradiente de Optiprep

foi preparado como previamente descrito (Cantin, et al. 2008). O gradiente foi preparado

variando de 18% a 6% em incrementos de 1.2%. Ao todo 11 frações de 300 µl foram diluídas em

PBS a partir da solução estoque de 60% de Optiprep. As vesículas foram aplicadas sobre o

gradiente e ultracentrifugadas em um rotor de ângulo móvel, SW50 (Beckman) a 250000xg por

75 minutos. Onze frações foram coletadas a partir da parte superior e cada fração foi analisada

quanto a presença de e esterol e GXM.

Para identificar vesículas produzidas durante a infecção in vitro de macrófagos, o sedimento

100000xg obtido como no protocolo descrito anteriormente e foi submetido a um gradiente de

sacarose. Após a purificação das vesículas, estas foram homogeneizadas em uma solução com

concentração de 85% (peso/volume) de sacarose em TBS, gerando uma concentração final de

42.5% de sacarose. Esta suspensão foi transferida para um tubo de ultracentrífuga e uma solução

de 35% de sacarose foi aplicada sobre a anterior, seguido de uma última camada de 5% de

sacarose em TBS. O gradiente foi centrifugado por 18h a 200000xg e frações de 0.25 ml foram

coletadas a partir do topo do gradiente. As frações foram extensivamente dialisadas contra TBS,

secadas por centrifugação a vácuo e preparadas para análise lipídica em HPLC e detecção de

GXM, conforme descrito acima.

M. Cultivo de macrófagos murinos:

As linhagens celulares de macrófagos murinos RAW 264.7 e J774.16 (obtidas a partir da

ATCC), foram cultivadas em meio mínimo essencial de Dulbecco (DMEM) suplementado com 10%

de soro fetal bovino (FCS), 2mM de L- glutamina, 1mM de piruvato de sódio, aminoácidos não-

essenciais (Gibco-Invitrogen cat. N. 11360), 10mM de HEPES e 50 mM de 2-β-mercaptoetanol. As

células foram mantidas a 37

C em atmosfera de 7.5% de CO

e sob condições livres de LPS.

N. Incorporação de componentes vesiculares por macrófagos:

Células RAW264.7 foram plaqueadas em placas de 24 poços contendo lamínulas na

proporção de 1x10

células por poço. Amostras de vesículas foram coradas com o fluoróforo

lipofílico dialquilcarbocianina (DiI) (Invitrogen, V22885). Suspensões de vesículas foram

normalizadas para 2 µg/ml de esterol e suplementadas com DiI em uma concentração final de

3µM. Após 30 minutos a temperatura ambiente, a suspensão de vesículas foi ultracentrifugada a

100000xg por 1h e lavada 3 vezes em PBS. As vesículas coradas com DiI foram então incubadas

com os macrófagos por 30 minutos a 37

C, 7.5% CO

. Como controle incubamos meio estéril

com DiI, ultracentrifugamos e lavamos 3 vezes, sob as mesmas condições que a suspensão de

vesículas. Os macrófagos foram então fixados com paraformaldeído 4% em PBS por 5 minutos a

C e bloqueados com PBS 5% BSA por 1 hora a temperatura ambiente. Para visualização da

membrane plasmática foi utilizada a subunidade B da toxina do cólera marcada com FITC (CtxB-

FITC), a qual foi incubada com as células bloqueadas por 1h na concentração de 1 µg/ml. CtxB-

FITC liga-se a GM1, gangliosídeo majoritariamente presente na membrane plasmática. As células

foram lavadas 3 vezes com PBS e o núcleo foi evidenciado com 4’,6-diamidino-2-fenilindol (DAPI)

a 10 µg/ml. As amostras foram montadas em lâminas contendo Meio de Montagem (50% de

glicerol e 50 mM de N-propil galato em PBS) e foram visualizadas em microscópio confocal Leica

AOBS com laser de varredura (Mannheim, Alemanha). Foi utilizada lente objetiva com aumento

de 63x em óleo de imersão. Imagens tridimensionais foram obtidas a partir da reconstrução de

eixo z utilizando o software ImageJ (NIH [http://rsb.info.nih.gov/ij/]). Alternativamente, células

foram incubadas com vesículas coradas com DiI, bloqueadas com PBS 5%BSA e incubadas por 1h

com soro de pacientes com criptococose. Como controle negativo foi usado soro de indivíduos

sadios. Os soros foram diluídos 100 vezes em PBS 1% BSA. Após 3 lavagens as amostras foram

incubadas com anticorpo anti- IgG humano conjugado a AlexaFluor488 (Invitrigen). As amostras

foram novamente lavadas, o núcleo foi corado com 10µg/ml de DAPI e lâminas foram montadas

em Meio de Montagem. As amostras foram visualizadas em um microscópio de fluorescência

Axioplan 2 (Zeiss, Alemanha), as imagens adquiridas usando a câmera digital Color View SX e

processadas com o software analySIS (Soft Image System).

O. Ensaio de Atividade de Lactato Desidrogenase (LDH):

Para avaliar se a exposição de macrófagos às vesículas gera dano celular, a atividade da

enzima citoplasmática LDH foi medida nos sobrenadantes de cultura. Células da linhagem

RAW264.7 foram plaqueadas na densidade de 1x10

células/ ml em placas de 24 poços e

estimuladas com suspensão de vesículas à 2µg/ml de esterol por 16h. Alíquotas do sobrenadante

foram coletadas e suplementadas com NADH e piruvato de sódio ajustados para concentrações

finais de 0.3 e 4.7 mM, respectivamente. Uma cinética de decaimento da absorbância a 340 nm

pelo NADH consumido foi feita do tempo 0 à 10 minutos em um espectrofotômetro. O controle

positivo foi gerado adicionando 10% de Triton X-100 às culturas e o controle negativo foi usado

sobrenadante de células não- estimuladas incubadas em meio por 16h. O experimento foi feito

em triplicatas e estatisticamente analisado pelo teste t Student’s.

P. Produção de citocinas e NO por macrófagos estimulados com vesículas:

Células RAW foram plaqueadas em placas de 96 poços a 1x105 células/ poço. As

monocamadas foram lavadas duas vezes com meio sem soro, o qual foi substituído por 1% de

Nutridoma-SP (Boehringer Mannheim), e incubadas com diferentes diluições de vesículas por

16h a 37

C, 7.5% CO

. Como controle positivo foi usado 1µg/ml de LPS. Os sobrenadantes foram

coletados e as citocinas interleucina 10 (IL-10), Fator de Transformação do Crescimento β (TGF-β)

e Fator de Necrose Tumoral α (TNF-α) foram testadas por ELISA (R&D systems). Os níveis de

óxido nítrico (NO) no sobrenadante foram medidos pelo método de Griess (Green, et al. 1982). O

inibidor específico da enzima NO- sintase induzida (iNOS), N6-(1-Iminoetil)-L-lisina dihidrocloreto

(L-NIL) (Cayman Chemical), foi usado para confirmar que a enzima é especificamente induzida

durante o processo de ativação descrito. Para estes experimentos as células foram estimuladas

com vesículas por 16h na presença ou ausência de 3µM de L-NIL. Para averiguar se a resposta

aos estímulos se dá pela presença de LPS contaminante, experimentos controles foram feitos na

presença de 10µg/ml de Polimixina B e a produção de NO foi avaliada. Todos os experimentos

foram feitos em triplicatas e analisados estatisticamente pelo ANOVA seguido de pos-teste de

Bonferroni.

Q. Determinação do índice de fagocitose por Citometria de Fluxo:

Para avaliar se estímulo com vesículas altera a capacidade fagocítica de macrófagos frente ao

C. neoformans, foi utilizado o protocolo baseado no método de Chaka (Chaka, et al. 1995). As

leveduras foram incubadas com isotiocianato de fruoresceína (FITC) a 0.5 mg/ml em PBS por 10

minutos. Os macrófagos foram previamente estimulados por 16h com preparações de vesículas

de diferentes cepas de C. neoformans na concentração de 1 µM de esterol. Após lavar

extensivamente com PBS, of fungos foram incubados com células RAW por 3h na proporção 5:1

fungo/ célula, seguido de sucessivas lavagens para retirar os fungos não aderidos. A intensidade

de fluorescência dos macrófagos é função direta da associação com fungos marcados com FITC.

As células infectadas foram então deaderidas, fixadas com PBS 4% paraformaldeído, lavadas e

analisadas em citômetro de fluxo FACScalibur (BD Biosciences). Os dados foram analisados

usando o software winMDI2.9 e estatisticamente validados pelo teste de variança ANOVA

seguido do pos-teste de Bonferroni. Para visualizar o aspecto geral das células, lamínulas de

células infectadas com fungos marcados com FITC foram preparadas em paralelo e observadas

por epifluorescência (Axioplan 2- Zeiss, Alemanha). As imagens foram adquiridas em câmera

digital Color View SX e processadas pelo software analySIS.

R. Determinação da atividade microbicida de macrófagos estimulados com vesículas:

Monocamadas de macrófagos foram estimuladas por 16h com preparações de vesículas de

diferentes cepas de C. neoformans em concentração correspondente a 0.4 µg/ml de esterol. O

sobrenadante foi removido e a monocamada foi lavada com DMEM sem soro. Células fúngicas

foram ressuspensas em DMEM e adicionadas as monocamadas celulares na razão 5:1

fungo/célula. Após incubação por 3h a 37

C, 7.5% CO

, as amostras foram lavadas 3 vezes em

PBS para remover os fungos não aderidos e meio fresco foi adicionado para continuar as

incubações por 2h ou 5h (total de 5h e 8h de incubação) a 37

C, 7.5% CO

. Após as incubações,

as células foram novamente lavadas e lisadas com H

O destilada gelada e estéril. Macrófagos

lisados contendo células fúngicas foram imediatamente plaqueados em agar Sabouraud para

contagem de UFC. Os controles consistem em macrófagos não estimulados. Todos os

experimentos foram feitos em triplicatas e analisados estatisticamente pelo teste de variança

ANOVA seguido de pos-teste de Bonferroni.

S. Análise do diâmetro de vesículas por Espalhamento de Luz:

Vesículas em suspensão permanecem em movimento Browniano, o qual pode ser traduzido

em flutuações no espalhamento de luz quando em fase líquida. Essa propriedade pode ser

medida pela técnica de Espalhamento Dinâmico de Luz (dynamic light scattering, DSL), e revela

informações sobre diâmetro e heterogeneidade da amostra (Eisenman, et al. 2009). Medidas de

tamanho de vesículas foram feitas em um analisador de partículas 90Plus/BIMAS Multi Angle

Particle Sizing (Brookhaven Instruments) como descrito previamente (Eisenman, et al. 2009).

T. Identificação de proteínas por LC- MS/MS:

A caracterização proteômica de vesículas de diferentes mutantes de S. cerevisiae foi feita

com base na metodologia recentemente descrita para análise de vesículas fúngicas

(Albuquerque, et al. 2008; Rodrigues, et al. 2008). As vesículas purificadas foram ressuspensas

em 40ml de NH4HCO3 a 400 mM contendo 8M de uréia e as pontes dissulfeto foram reduzidas

através da adição de 10ml de ditiotreitol (DTT) a 50 mM, seguido de incubação por 15 minutos a

50 C. Resíduos de cisteína foram alquilados pela adição de 100 mM de iodoacetamina (10 ml),

seguido de 15 minutos de incubação a temperatura ambiente sob proteção da luz. A

concentração final de uréia foi ajustada para 1M e a mistura foi suplementada com 4 mg de

tripsina (Promega). A digestão acorreu por 16h a 37

C. Os peptídeos resultantes foram

submetidos a cromatografia de fase reversa em colunas C18 (POROS R2 50, Applied Biosystems).

Para isto, as amostras foram homogeneizadas em 5% de acetonitrila (ACN) contendo 0.5% de

ácido fórmico (FA) e foram por fim aplicadas as colunas C18. Os peptídeos foram eluídos em um

gradiente linear (5-40% CAN/0.1%FA) durante 200 minutos e diretamente analisados por ESI-MS

(LTQXL/ETD, Thermo Fisher). O Nanospray foi fixado a 1.35 kV em uma pressão de 0.2 psi N

. Os

espectros foram coletados entre 400-1700 m/z e os 10 íons mais abundantes foram submetidos

por duas vezes a dissociação induzida por colisão (CID), com energia de colisão normalizada para

35%. Todos os espectros MS/MS de peptídeos entre 800-3500 Da, com mais de 10 contagens e

com pelo menos 15 fragmentos foram convertidos em arquivos DTA utilizando o programa

Bioworks v. 3.3.1 (ThermoFisher). Arquivos DTA foram submetidos a pesquisa usando

TurboSequest (Eng, et al. 1994) e base de dados de proteínas de S. cerevisiae

(www.yeastgenome.org) combinado a análise de sequencias de queratina humana e tripsina

(www.ncbi.nlm.nih.gov/protein). Proteínas assinaladas foram re-filtradas com total de Xcorr $

3.5. A taxa de falso positivo (false-discovery rate, FDR) foi estimada como descrito previamente

(Liu, et al. 2004). Proteínas com peptídeos em comum foram agrupadas para avaliar o índice de

redundância. Determinação semi-quantitativa da abundância relativa de proteínas foi feita com

base na contagem spectral de cada proteína (Liu, et al. 2004). Este método foi ainda validado

pelo cálculo do índice de abundância de proteína modificado exponencialmente (emPAI), o qual

foi deduzido segundo a metodologia proposta por Ishihama et. al. (Ishihama, et al. 2005). Foi

usado cutoff de 10 espectros para validar assinalações positivas, resultando em um total de 127

proteínas sequenciadas e validadas.

U. Sistemas de predição e análise de interações funcionais baseadas nos dados de

proteoma:

Diagramas de Venn foram construídos utilizando a ferramenta Venny, disponível em

http://bioinfogp.cnb.csic.es/tools/venny/index.html. Interações funcionais entre proteínas foram

caracterizadas usando o programa Osprey Network Visualization (version 1.2.0), utilizando a base

de dados de S. cerevisiae disponibilizada pelo próprio software. Análise de predição de proteínas

contendo possíveis sítios de ligação a âncoras de GPI foram feitas pelo programa FragAnchor

(http://navet.ics.hawaii.edu/,fraganchor/NNHMM/NNHMM.html) (Poisson, et al. 2007). A

predição de proteínas contendo sítio de clivagem do peptídeo sinal para síntese no retículo

endoplasmático (RE) foi feita através do programa SignalP 3.0

(http://www.cbs.dtu.dk/services/SignalP/), conforme descrito previamente (Bendtsen, et al.

2004).

VI – Resultados:

A seção de resultados apresentados nessa tese consiste em dados publicados em 4 artigos

científicos em revistas revisadas internacionalmente. Estes trabalhos encontram-se em anexo

(Anexos 1 a 4) e serão mencionados no decorrer do texto. Esclarece-se, portanto, que a descrição

abaixo corresponde a uma versão resumida dos resultados, cuja descrição detalhada está

disponível nas publicações citadas (Anexos).

1) A secreção de polissacarídeo capsular em vesículas é uma solução eucariótica para o

problema do transporte de macromoléculas através da parede celular.

Evidências prévias da literatura sugeriam que o polissacarídeo capsular de C. neoformans,

bem como o esfingolipídio imunogênico GlcCer, poderiam ser transportados para o espaço

extracelular em carreadores vesiculares (Rodrigues, et al. 2000; Sakaguchi, et al. 1993). O

presente trabalho (Anexo 1) comprovou pela primeira vez a existência de vesículas extracelulares

secretadas por um fungo.

Vesículas em associação com a parede celular puderam ser observados por TEM (Figura 1A-C,

Anexo 1). Estruturas vesiculares também foram observadas por TEM durante infecção in vivo, em

cortes de tecido pulmonar de camundongos infectados (Figura 1, D e E). Estas micrografias

sugeriram que vesículas eram capazes de atravessar a parede celular, e portanto deveriam ser

encontradas no espaço extracelular. Para testar tal hipótese, foi utilizado um protocolo clássico

de isolamento de exossomos baseado em etapas de centrifugações diferenciais e

ultracentrifugações. O sedimento originado da ultracentrifugação a 100000xg foi submetido a

preparação para TEM. As preparações mostraram se tratar de vesículas dotadas de bicamada

lipídica, relativamente heterogêneas em termos de tamanho e eletrondensidade, como pode-se

observar na Figura 1 F-H.

A composição lipídica dessas vesículas foi analisada por HPTLC e por ESI-MS/MS. Foi possível

detectar a presença dos dois esteróis majoritários do C. neoformans, ergosterol e obtusifoliol,

bem como do principal cerebrosídeo do C. neoformans, N-2’-hidroxioctadecanoil-1-β-D-

glucopiranosil-9-metil-4,8-esfingadienina (Anexo 1, Figura 2). Fosfatidilcolina (PC) é o principal

fosfolipídio constituinte de membranas no C. neoformans (Rawat et al., 1984). Por isso,

analisamos amostras de vesículas por LC-MS/MS operando no modo de busca do íon precursor,

ajustado para o fragmento 184.1 m/z, o qual corresponde a PC. Foi possível identificar três

diferentes espécies de PC que variam quanto a composição do ácido graxo, sendo estas:

P (782.570 Da); C

P (784.586 Da); C

P (786.601 Da) (Anexo 2, Figura

4B). Para garantir que a caracterização lipídica não incluiria frações de membranas não-

vesiculares, controles de integridade vesicular foram feitos por TEM. Vesículas apresentaram

bicamada lipídica e tamanho variando de 50 a 300 nm, como estabelecido previamente (Anexo 2,

Figura 4A).

Foi visto também que a secreção vesicular é dependente de viabilidade celular, sendo

portanto um fenômeno biológico ativo. Mesmo apresentando mais de 99% de viabilidade celular

após 72h de cultivo, era necessário comprovar que as vesículas não eram oriundas da minoria de

células mortas. Para isso, foi desenvolvido um experimento de pulso e caça, utilizando

precursores radioativos da ceramida uma vez que GlcCer foi caracterizada como marcador de

vesículas (Anexo 1, Figura 2 e Figura 3B). A detecção de [

H]GlcCer nas frações vesiculares indica

que este mecanismo de secreção requer células metabolicamente ativas (Anexo 1, FiguraFigura

3A). Ainda assim as vesículas poderiam ser oriundas de extravasamento citoplasmático de

organelas, uma vez que a [

H]GlcCer seria incorporada na maioria das membranas intracelulares.

Para excluir essa hipótese, o perfil lipídico de vesículas isoladas sob condições normais de cultivo

foi comparado ao perfil de vesículas isoladas de células mortas. Vesículas foram isoladas do

sobrenadante de cultura de células mortas por inibição metabólica (10mM de NaN

), onde se

observou 83% de morte celular, ou por aquecimento (1h, 50

C), método que resultou em 99%

de morte celular. Em ambos os casos observou-se uma redução expressiva da quantidade de

lipídeos no sedimento 100000xg (Anexo 1, Figura 3C).

Para avaliar se as vesículas lipídicas carreariam o polissacarídeo formador de cápsula, foram

feitos imunoensaios para detecção de GXM no sedimento obtido por centrifugação a 100000xg

com o anticorpo monoclonal anti-GXM Mab18B7. Foi demonstrado por imuno-microscopia

eletrônica de transmissão, bem como por ELISA, que de fato a GXM se encontra no interior das

estruturas vesiculares (Anexo 1, Figura 4A e 4B). Para confirmar que a GXM detectada por ELISA

era de fato intravesicular, a amostra de vesículas foi sucessivamente passada por uma coluna de

afinidade acoplada ao Mab18B7. Alíquotas foram retiradas após cada passagem e observou-se

por ELISA que os níveis de GXM mantiveram-se constantes, enquanto os níveis de GXM livre

purificada caem em cada passagem (Anexo 1, Figura 4C). Sobrenadantes de cultura de C.

neoformans são extremamente ricos em GXM livre. Para excluir a possibilidade de vesículas

formarem-se randomicamente pela agregação de lipídeos liberados no sobrenadante de cultura

e desta maneira englobarem a GXM livre, vesículas foram isoladas do mutante acapsular Cap67

em diferentes condições. Este experimento baseou-se no fato de que o Cap67 é capaz de

produzir vesículas, entretanto estas não contém GXM. O mutante acapsular foi crescido em meio

mínimo, meio mínimo suplementado com GXM e em uma terceira condição o meio foi

suplementado com GXM após o cultivo celular. Em ambas as condições a GXM adicionada não foi

encontrada na fração vesicular (Anexo 1, Figura 4D).

Foi visto também que há uma correlação direta entre o tamanho da cápsula e a secreção

vesicular de GXM. Uma vez que a cápsula foi induzida, observou-se por ELISA um concomitante

aumento do conteúdo vesicular de GXM (Anexo 1, Figura 5). Além disso, pode-se observar que o

mutante acapsular foi capaz de reincorporar a GXM vesicular (Anexo 1, Figura 6), formando uma

cápsula evidenciada por imunofluorescência. Estes resultados agregam função biológica ao

mecanismo de secreção vesicular.

2) Crioultramicrotomia do C. neoformans e fracionamento vesicular revelaram associação

íntima entre lipídeos e GXM.

Protocolos para IEM (immunoelectron microscopy) em células fúngicas em geral envolvem

etapas como degradação enzimática da parede celular que podem gerar artefatos, os quais

limitam a interpretação das observações (Yamaguchi, et al. 2005). A fim de obter preparações

preservadas de C. neoformans para IEM, aplicamos o protocolo de crioultramicrotomia. Foi

possível obter preparações extremamente bem preservadas, sobretudo em relação as organelas

membranosas e a parede celular (Anexo 2 Figura 1). Inclusive compartimentos nunca antes

descritos em C. neoformans foram visualizados por esta técnica, como vesículas citoplasmáticas

semelhantes a endossomos e organelas eletrondensas semelhantes a acidossomos (Anexo 2,

Figura 1B e 2).

A marcação intracelular para imuno-microscopia eletrônica de transmissão com Mab18B7 foi

na maioria das vezes encontrada em associação com bicamadas lipídicas (Anexo 2, Figura 3). Tais

estruturas membranosas assemelham-se a vesículas carreadoras de GXM, corpos multi-

vesiculares e membranas reticulares posicionadas na periferia celular (Anexo 2, Figura 3). Neste

estudo, foi possível observar tanto a presença de vesículas intracelulares contendo GXM quanto

de vesículas intracelulares não contendo GXM (Anexo 2, Figura 1C, 2). Nesse contexto,

resolvemos avaliar se a distribuição de GXM nas vesículas extracelulares obedecia a mesma

regra. O fracionamento das vesículas em gradiente de densidade revelou a existência de

subpopulações de vesículas que contém e que não contém GXM (Anexo 2, Figura 5). Esta

heterogeneidade de conteúdo sugere a existência de mais de uma via intracelular de produção

de vesículas.

3) Vesículas extracelulares secretadas pelo C. neoformans modulam a função de

macrófagos.

A fim de entender a influência da secreção de vesículas no processo de infecção celular,

primeiramente foi demonstrado que vesículas são produzidas durante a infecção in vitro de

macrófagos. A interação de fungos opsonizados com Mab18B7 ocorreu por 1h na proporção 5:1

fungo: célula. Após a lavagem, as monocamadas de células J774 foram incubadas por 16h e

então, após a confirmação microscópica da lise celular, os sobrenadantes de cultura foram

recolhidos e avaliados para presença de componentes vesiculares. Devido a maior complexidade

do sistema, as vesículas foram fracionadas em gradiente de sacarose e frações contendo GlcCer e

GXM foram detectadas (Anexo 1, Figura 7).

Para avaliar se macrófagos incorporam componentes vesiculares, as células foram incubadas

com vesículas isoladas e marcadas com o fluoróforo DiI, o qual intercala-se em membranas. Foi

visto por microscopia confocal que membranas vesiculares se encontram no interior dos

macrófagos após 30 minutos de incubação (Anexo 3, Figura 1A). Além das membranas, proteínas

imunogênicas sabidamente presentes nas vesículas (Rodrigues, et al. 2008) também se

encontram no interior de macrófagos em co-localização com a marcação de DiI (Anexo 3, Figura

1B). Foi demonstrado também, através do ensaio de atividade de LDH, que a despeito da

presença de diversos fatores de virulência nas vesículas (Rodrigues, 2008), estas não causam lise

celular (Anexo 3, Figura 1C).

Este trabalho demonstrou que vesículas secretadas pelo C. neoformans são capazes de

modular a produção de NO e citocinas por células RAW (Anexo 3, Figura 2 e 3). A composição

vesicular, sobretudo no que tange ao conteúdo de GXM, tem influência direta sobre quais

citocinas serão elevadas ou diminuídas e sobre a intensidade de produção de NO (Anexo 3,

Figura 2 e 3). A atividade fagocítica de macrófagos estimulados com vesículas bem como a

atividade microbicida também são significativamente aumentados (Anexo 3, Figura 4 e 5).

Fungos marcados com FITC foram usados num ensaio de citometría de fluxo para quantificar o

índice de fagocitose. Observou-se um aumento relativo de até 74% na população de macrófagos

positivos para FITC quando os fagócitos foram pré-estimulados com vesículas (Anexo 3, Figura

4A). Microscopias de fluorescência da mesma amostra confirmaram essa observação, uma vez

que macrófagos estimulados com vesículas apresentaram um número evidentemente maior de

fungos internalizados (Anexo 3, Figura 4B). Após 5h e 8h de infecção, foi quantificado o número

de fungos viáveis no interior de macrófagos através da técnica de UFC. Foi possível concluir que

macrófagos pré-incubados com vesículas isoladas apresentaram aumento da capacidade

fungicida e mais do que isso, essa aumento foi ainda mais expressivo quando as células foram

tratadas com vesículas de CAP67, as quais não contém GXM (Anexo 3, Figura 5).

4) Caracterização de vesículas extracelulares de S. cerevisiae: evidencias para participação

de diferentes vias de tráfego na biogênese vesicular.

S. cerevisiae foi a levedura escolhida como modelo de estudo uma vez que é o organismo em

que mais se compreende os mecanismos de tráfego intracelular. Mutantes com defeitos em

diferentes pontos das vias de tráfego foram selecionados com base em dados prévios da

literatura e vesículas foram isoladas e caracterizadas a partir destas diferentes cepas listadas na

Tabela 1 do anexo 4. Primeiramente foi feita a caracterização das vesículas quanto ao tamanho

por Espalhamento de Luz Dinâmico e quanto ao aspecto morfológico por TEM (Anexo 4, Figura

1). Foram isoladas vesículas de dois mutantes de vias de tráfego, o sec4-2 ts e o snf7



. Apenas o

mutante sec4-2 ts apresentou alteração significativa do diâmetro vesicular (Anexo 4, Figura 1B)

enquanto na TEM de vesículas ambos os mutantes mostraram-se morfologicamente semelhantes

ao tipo selvagem (WT) (Anexo 4, Figura 1A).

Figura 11. Etapas do tráfego intracelular bloqueadas por diferentes mutações. Cinco mutantes foram

utilizados no estudo de proteômica. O mutante bos1-1 ts acumula vesículas que brotam do RE. O mutante

sec4-2 ts acumula vesículas que brotam da TGN. O mutante sec1-1 ts acumula vesículas estágio de fusão

com a membrane plasmática. O mutante vps23



apresenta defeito no endereçamento de moléculas para

os MVBs. O mutante snf7



apresenta problemas na formação de vesículas internas dos MVBs.

A análise proteômica das vesículas de S. cerevisiae foi feita com 3 cepas selvagens e 5

diferentes mutantes (Anexo 4, Tabela 1 e Figura 11). Um resumo das proteínas encontradas,

classificadas por função biológica encontra-se na Figura 2A bem como na Tabela S1 do anexo 4.

Um universo tão diverso de proteínas encontradas dentro de um mesmo compartimento pode

parecer contraditório. Entretanto 90% das proteínas caracterizadas apresentam algum tipo de

interação funcional com alguma outra dentro do proteoma vesicular (Anexo 4, Figura 2B). A

caracterização proteômica incluiu estudos de predição do sítio de clivagem do peptídeo sinal

para síntese no RE e de sequencias para ancoramento de GPI. De acordo com esses dados de

bioinformática, 4% das proteínas apresentam seqüência para ancoramento de GPI, contra 1% no

proteoma total da célula. Também observou-se uma concentração de aproximadamente 4 vezes

de proteínas sintetizadas no RE em comparação com o proteoma total da célula (Anexo 4, Figura

3). O perfil qualitativo do proteoma não variou significativamente entre células selvagens e

mutantes, como representado nos diagramas de Venn (Anexo 4, Figura 4). O mutante snf7



foi o

que apresentou a maior alteração (72% de similaridade com o WT). Diante disso, resolvemos

avaliar se proteínas individuais seriam alteradas quantitativamente. Para tanto, calculamos o

valor de emPAI (exponentially modified protein abundance index) para cada proteína de cada

amostra. Dentre estas, 57.5% das proteínas foram significativamente alteradas em sua

abundância, as quais estão listadas na Tabela S2 do anexo 4. Gráficos de correlação comparando

a abundância relativa de proteínas de cada mutante com seu respectivo WT foram montados e o

cálculo da regressão linear revelou que o mutante snf7



é o que mais se difere do WT em

relação a composição proteômica das vesículas (Anexo 4, Figura 5).

Análises das frações lipídicas dos mutantes sec4-2 ts e snf7



mostraram que há uma redução

expressiva da secreção total de vesículas no mutante sec 4-2 ts, enquanto que no mutante snf7



a secreção não tenha sido quantitativamente alterada (Anexo 4, Figura 6A e B). O mutante sec 4-

2 ts acumula esterol no interior celular (Anexo 4, Figura 6C), o que está de acordo com sua

capacidade diminuída de liberar vesículas contendo esterol. A diminuição da secreção de

vesículas no mutante sec 4-2 ts foi evidente após o acompanhamento da cinética de secreção a

qual se estagnou após 3h no mutante enquanto que no WT teve o pico de secreção em 18h. A

análise de um outro mutante envolvendo mecanismos de secreção não-convencional (Figura 7

do Anexo 4) revelou que a deleção do gene de GRASP causa redução no conteúdo de esterol das

frações vesiculares e acúmulo intracelular de esterol, assim como visto para o mutante sec4-2 ts.

Anexo 1:

EUKARYOTIC CELL, Jan. 2007, p. 48–59 Vol. 6, No. 1

1535-9778/07/$08.00ϩ0 doi:10.1128/EC.00318-06

Vesicular Polysaccharide Export in Cryptococcus neoformans Is a

Eukaryotic Solution to the Problem of Fungal Trans-Cell

Wall Transport

ᰔ

Marcio L. Rodrigues,

‡ Leonardo Nimrichter,

‡De´bora L. Oliveira,

‡ Susana Frases,

Kildare Miranda,

Oscar Zaragoza,

† Mauricio Alvarez,

Antonio Nakouzi,

Marta Feldmesser,

2,4

and Arturo Casadevall

2,4

Laborato´rio de Estudos Integrados em Bioquı´mica Microbiana, Instituto de Microbiologia Professor Paulo de Go´es,

Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941590, Brazil

; Department of Microbiology and

Immunology

and Division of Infectious Diseases

of the Department of Medicine, Albert Einstein College of

Medicine, 1300 Morris Park Ave., Bronx, New York 10461; and Laborato´rio de

Ultraestrutura Celular Hertha Meyer, Instituto de Biofı´sica Carlos Chagas Filho,

Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941590, Brazil

Received 6 October 2006/Accepted 6 November 2006

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A

central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the

mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular

environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular

vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the

extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the crypto-

coccal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing

vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major

virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the

capsule network to reach the extracellular environment.

Cryptococcus neoformans is a yeast-like pathogenic fungus that

is the etiologic agent of human cryptococcosis. Infection is usually

asymptomatic and restricted to the lung in immunocompetent

individuals, but fungal cells can disseminate to other organs and

cause cryptococcal meningoencephalitis, a common syndrome in

immunosuppressed patients (29, 39). Signiﬁcant progress in our

understanding of how C. neoformans causes disease has been

made in the last decade, but many aspects of cryptococcal patho-

genesis remain poorly understood.

C. neoformans represents a unique model in cell biology stud-

ies because it is the only eukaryotic pathogen with a polysaccha-

ride capsule, a structure that is essential for virulence (4, 19). The

major capsular polysaccharide glucuronoxylomannan (GXM) has

an average mass ranging from 1.7 ϫ 10

to 7 ϫ 10

daltons (22)

and is released extracellularly during infection, inducing a number

of deleterious effects to the host (39). GXM also represents a

potential vaccine component and is the target of therapeutic an-

tibodies that are currently in clinical trial (3, 8, 20, 27).

The fungal cell wall is a compact, albeit dynamic, structure that

plays important roles in several biological processes that deter-

mine cell shape, morphogenesis, reproduction, cell-cell and cell-

matrix interactions, and osmotic and physical protection (25).

Although the relevance of the cell wall to fungal physiology and

pathogenesis is clear, the mechanisms by which large molecules

(molecular weight, Ͼ1 million) cross this rigid structure to reach

the extracellular environment are largely unknown. In C. neofor-

mans, it has been suggested that capsule components containing

epitopes recognized by a monoclonal antibody (MAb) to GXM

are synthesized intracellularly and exported through the cell wall,

possibly inside membrane vesicles (11). These results were vali-

dated in a recent study using a secretion mutant of C. neoformans

in which post-Golgi secretory vesicles containing GXM were ac-

cumulated in the plasma membrane region (40). Based on these

observations, one could imagine that surface components of C.

neoformans, including the capsule, are synthesized in the cyto-

plasm and exported to the exterior of the cell in secretory vesicles

that traverse the cell wall. Indeed, C. neoformans does produce

glucosylceramide (GlcCer)-containing vesicles that are trans-

ferred to the cell wall (1, 25, 30). Since C. neoformans efﬁciently

releases exocellular capsular material, we hypothesized the exis-

tence of extracellular secreted vesicles containing GlcCer and

capsule components.

In the present work, we describe for the ﬁrst time that a fungal

cell can produce extracellular vesicles that are secreted across the

cell wall. Supernatants of C. neoformans cultures contained vesi-

cles with bilayered membranes. Lipid analysis revealed that key

fungal lipids, such as GlcCer, ergosterol, and a novel sterol, are

present in these membranes. By different approaches, we dem-

onstrated that GXM is packaged inside the vesicles, which cross

the cell wall and the capsule network to reach the extracellular

environment. A correlation between capsule growth and detec-

* Corresponding author. Mailing address: Department of Microbi-

ology and Immunology, Albert Einstein College of Medicine, 1300

Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-2215. Fax:

(718) 430-8968. E-mail: [email protected].

† Present address: Servicio de Micologı´a, Centro Nacional de Mi-

crobiologı´a, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo

km 2, 28220 Majadahonda, Madrid, Spain.

‡ M.L.R., L.N., and D.L.O. contributed equally to this work.

ᰔ

Published ahead of print on 17 November 2006.

tion of vesicle-associated GXM was observed, suggesting that C.

neoformans can release the polysaccharide from the vesicles and

incorporate it into the cell surface. Accordingly, acapsular cells

used vesicle-associated GXM to become encapsulated. GXM-

containing vesicles were produced during macrophage infection,

suggesting a role in pathogenesis. These ﬁndings illustrate a new

phenomenon in fungi with potential relevance for such diverse

areas as capsule assembly and pathogenesis and reveal new in-

sights into how secreted molecules reach the extracellular envi-

ronment.

MATERIALS AND METHODS

Culture conditions. The cryptococcal isolates used in this study comprised

strains ATCC 24067 (serotype D; American Type Culture Collection), H99

(serotype A; clinical isolate), HEC3393 (serotype A; clinical isolate), and Cap67

(acapsular mutant). In the different analyses performed, similar data were ob-

tained with encapsulated strains. Except for the presence of GXM, results for

assays using acapsular cells followed the same proﬁle obtained with HEC3393,

H99, and ATCC 24067 cells. For vesicle puriﬁcation, C. neoformans cells were

inoculated into 1,000-ml Erlenmeyer ﬂasks containing 400 ml of a minimal

medium composed of dextrose (15 mM), MgSO

(10 mM), KH

(29.4 mM),

glycine (13 mM), and thiamine-HCl (3 ␮M). Fungal cells were cultivated for 3

days at 30°C with shaking. The viability of C. neoformans cells after this period

of cultivation was analyzed by propidium iodide staining.

Isolation of vesicles. Fungal cells were separated from culture supernatants by

centrifugation at 4,000 ϫ g for 15 min at 4°C. The supernatants were collected

and again centrifuged at 15,000 ϫ g (4°C) to remove smaller debris. The pellets

were discarded, and the resulting supernatant was concentrated approximately

20-fold using an Amicon ultraﬁltration system (cutoff, 100 kDa). To ensure the

removal of cells and cellular debris, the concentrated culture ﬂuid was again

centrifuged as described above and the resulting supernatant was then centri-

fuged at 100,000 ϫ g for1hat4°C. The supernatants were then discarded, and

the pellets were washed by ﬁve sequential resuspension and centrifugation steps,

each consisting of 100,000 ϫ g for1hat4°Cwith 0.1 M Tris-buffered saline

(TBS). The pellets were then resuspended in ﬁxative solution for electron mi-

croscopy analysis. Alternatively, 100,000 ϫ g pellets were fractionated by afﬁnity

chromatography and sucrose density centrifugation or extracted with organic

solvents as detailed below.

TEM. Transmission electron microscopy (TEM) was used to visualize vesicles

isolated from supernatants and those that were cell associated, in vitro and in

vivo. The pellets obtained after washing and centrifugation at 100,000 ϫ g were

ﬁxed in 2% glutaraldehyde in 0.1 M cacodylate at room temperature for2hand

then incubated overnight in 4% formaldehyde, 1% glutaraldehyde, and 0.1%

phosphate-buffered saline (PBS). The samples were incubated for 90 min in 2%

osmium, serially dehydrated in ethanol, and embedded in Spurr’s epoxy resin.

Thin sections were obtained on a Reichert Ultracut and stained with 0.5% uranyl

acetate and 0.5% lead citrate. Samples were observed in a JEOL 1200EX trans-

mission electron microscope operating at 80 kV. For immunogold labeling with

antibodies to GXM, the vesicles were ﬁxed in 0.1 M sodium cacodylate buffer

(pH 7.2) containing 4% paraformaldehyde, 0.2% glutaraldehyde, and 1% picric

acid, inﬁltrated in 25% polyvinylpyrrolidone and 2.1 M sucrose, and rapidly

frozen by immersion in liquid nitrogen. Cryosections were obtained in a tem-

perature range of Ϫ70 to Ϫ90°C using an Ultracut cryo-ultramicrotome

(Reichert). After being blocked in PBS-bovine serum albumin and 50 mM

Cl, the cryosections were incubated overnight in the presence of a mouse

monoclonal antibody to GXM (MAb 18B7, 1 ␮g/ml). The antibody is a mouse

immunoglobulin G1 (IgG1) with high afﬁnity for GXM of different cryptococcal

serotypes and has been extensively characterized previously (3). After incubation

with 15-nm (particle size) immunogold-labeled anti-mouse IgG, specimens were

observed in a JEOL 1200EX transmission electron microscope operating at 80 kV.

For detection of cryptococcal vesicles in vivo, murine infection and lung tissue

preparation for transmission electron microscopy were carried out as described

previously (10, 12, 26). Brieﬂy, anesthetized C57BL/6 mice (National Cancer

Institute, Bethesda, MD) were infected intratracheally via a midline neck inci-

sion with 10

or 10

organisms of C. neoformans strain ATCC 24067 and then

sacriﬁced 2 h, 48 h, or 7 days after infection. The analysis of vesicle production

in vitro was performed using strain Cap67 as previously described (30). Human

antibodies to GlcCer were used for lipid detection at the cell wall following

previously described experimental conditions (30).

Removal of nonvesicular GXM from 100,000 ؋ g pellets. To remove extrave-

sicular GXM and putative aggregates from vesicle preparations, 100,000 ϫ g pellets

were puriﬁed by passage through a column packed with an antibody-bound resin.

MAb 18B7 was coupled to cyanogen bromide-activated Sepharose according to the

manufacturer’s protocols (Sigma, Richmond, CA). Brieﬂy,1goftheresin was

suspended in 1 mM HCl, washed, and resuspended in carbonate buffer, pH 8.0.

MAb 18B7 was dissolved in 5 ml of the same buffer, at 1 mg/ml, and incubated

overnight with the resin. At this pH, MAb 18B7 retained its biological properties,

such as antigen binding and integrity of chains (not shown). Coupling efﬁciency was

ϳ90%, as conﬁrmed by spectrophotometric determinations at 280 nm. After being

washed with carbonate buffer and blocked with 0.2 M glycine, the resin was sequen-

tially washed with 0.1 M acetate (pH 4.0) and Tris (pH 8.0) buffers for ﬁnal resus-

pension in PBS. Each vesicle suspension (500 ␮l) was mixed with the antibody-

containing resin (100 ␮l) and incubated for 1 h (37°C) with shaking. The unbound

fraction was recovered by centrifugation. This process was repeated three times, and

the GXM contents in different fractions were analyzed by capture enzyme-linked

immunosorbent assay (ELISA) as described below.

Sucrose gradient. Sucrose density centrifugation was performed as described by

Gutwein et al. (15). The vesicle suspension (0.5 ml) obtained after ultracentrifuga-

tion and puriﬁcation with the antibody-coupled resin was mixed with an equivalent

volume of 85% (wt/vol) sucrose (in TBS), generating a ﬁnal concentration of 42.5%

sucrose. This suspension was transferred to an ultracentrifuge tube, and a step

gradient was prepared by overlaying the original suspension with 35% sucrose,

followed by a ﬁnal layer of 5% sucrose in TBS. The gradient was centrifuged for 18 h

at 200,000 ϫ g, and fractions of 0.25 ml were collected from the top to the bottom

of the gradient. Fractions were then extensively dialyzed against TBS, dried under

vacuum centrifugation, and suspended in 100 ␮l of absolute methanol, resulting in

the immediate formation of a precipitate. The solution was then extracted with 200

␮l of chloroform for1hat25°C. The organic fraction was recovered by centrifuga-

tion and analyzed by different methods for lipid identiﬁcation. The residual material

that was not soluble in the chloroform-methanol mixture was then dried under a

nitrogen stream, resuspended in TBS, and assayed for the presence of GXM, as

described later in this section.

Lipid analysis. The pellets obtained from centrifugation of cell supernatants at

100,000 ϫ g were ﬁrst suspended in methanol, and then two volumes of chloro-

form were added. The mixture was vigorously vortexed and centrifuged to dis-

card precipitates, dried by vacuum centrifugation, and partitioned according to

Folch et al. (13). The lower phase, containing neutral lipids, was recovered for

analysis by high-performance thin-layer chromatography (HPTLC). For sterol

analysis, the lipid extract was loaded into HPTLC silica plates (Si 60F254s;

LiChrospher, Germany) and separated using a solvent system containing hexane-

ether-acetic acid (80:40:2, vol/vol/vol) solvent. The plate was sprayed with a

solution of 50 mg ferric chloride (FeCl

) in a mixture of 90 ml water, 5 ml acetic

acid, and 5 ml sulfuric acid. After being heated at 100°C for 3 to 5 min, the sterol

spots were identiﬁed by the appearance of a red-violet color. The presence of

GlcCer was evaluated by separating lipids in Folch’s lower phase by HPTLC

using chloroform-methanol-water (65:25:4, vol/vol/vol) (13). Monohexosylcer-

amides were visualized after the plates were sprayed with orcinol-sulfuric acid

reagent, followed by heating at 150°C (1).

The lower phase obtained after Folch’s partition was also analyzed by elec-

trospray ionization mass spectrometry (ESI-MS) using a Finnigan LCQ DUO

ion trap instrument (Thermo Electron, San Jose, CA). Samples were diluted in

chloroform-methanol (1:1, vol/vol), containing 10 mM lithium iodide, and intro-

duced into ESI-MS at a 10-␮l/min ﬂow rate with the assistance of an infusion

micropump (Harvard Apparatus, Cambridge, MA). Analyses were carried out in

the positive mode. The source and capillary voltages were 4.5 kV and 3 V,

respectively. The capillary temperature was kept at 200°C. Spectra were collected

at a 300- to 800-m/z range. Source-induced dissociation was obtained at 25 V. Ion

trap collision-induced dissociation (ESI-MS/MS) experiments were carried out at

20 to 60% (1 to 3 eV) normalized relative collision energy. All spectra were

processed using Xcalibur software (Thermo Electron).

Lipids in fractions from the sucrose gradient were analyzed by high-perfor-

mance liquid chromatography (HPLC) in a Waters 600 liquid chromatograph

(New York, NY), using an Alltech C

column (Deerﬁeld, IL) (250- by 4.6-mm

dimensions, 5-␮m particle size). A mixture of chloroform and methanol (9:1,

vol/vol) was used as the mobile phase, and lipid components were detected by

absorbance at 280 nm in a Waters 486 detector. The proﬁle of elution of lipids

from sucrose gradient fractions was compared with a standard of puriﬁed cryp-

tococcal GlcCer (30).

Radioactive labeling of vesicles. C. neoformans (strain ATCC 24067) was

grown in the presence of

L-[

H]serine (20 Ci/mmol) (American Radiolabeled

Chemicals, Inc., St. Louis, MO) and [9,10-

H]palmitic acid (50 Ci/mmol)

(DuPont NEN, Boston, MA). These ceramide precursors were added to the

VOL. 6, 2007 GXM-CONTAINING VESICLES IN C. NEDFORMANS 49

culture medium described in “Culture conditions” to generate ﬁnal amounts of

radioactivity corresponding to 2.0 ␮Ci/ml ([

H]serine) and 1.0 ␮Ci/ml

([

H]palmitic acid). The culture was incubated for 3 days at 30°C, and then

fungal cells were removed by centrifugation. Vesicles were obtained as described

above and suspended in methanol (100 ␮l). Chloroform (200 ␮l) was added, and

the mixture was centrifuged to discard precipitates. The lipid extract was loaded

into HPTLC plates and separated using chloroform-methanol-water (65:25:4,

vol/vol/vol) as the solvent system. Molecules with migration rates corresponding

to that of GlcCer, identiﬁed by comparison with an iodine-stained standard of

glucocerebroside (Avanti Polar Lipids, Alabaster, AL), were scraped off the

plate, suspended in scintillation liquid, and counted for radioactivity. Negative

controls included 100,000 ϫ g pellets from sterile medium containing the radio-

active precursors and vesicle-depleted culture supernatants.

Fungal killing and lipid detection. To evaluate whether cryptococcal vesicles

were physiologically released or were simply an artifact of dead cells, we evalu-

ated the presence of lipid markers in 100,000 ϫ g pellets of living and dead cells.

Cryptococci were treated with 10 mM sodium azide in PBS for 60 min at 25°C or,

alternatively, suspended in PBS and heated at 50°C for the same period. Control

cells (viable) were suspended in PBS and incubated at 25°C for 60 min. Cell

viability was evaluated by inoculating control or treated yeasts onto Sabouraud

dextrose agar and counting CFU. After incubation under the conditions de-

scribed above, yeasts were removed as described previously in this section and

the supernatants ultracentrifuged at 100,000 ϫ g. The resulting pellets were

extracted with mixtures of chloroform-methanol at 2:1 (for GlcCer analysis) or

9:1 (for sterol analysis). Extracts were analyzed by HPTLC following the condi-

tions described in “Lipid analysis.”

Serological assays for GXM detection. The proﬁles of GXM distribution in the

fractions obtained after sucrose density centrifugation were analyzed using a

capture ELISA (2). Brieﬂy, each well of a 96-well polystyrene plate was coated

with a goat anti-mouse IgM. After removal of unbound antibodies, a solution of

MAb 12A1, an IgM MAb with speciﬁcity for GXM, was added to the plate, and

this step was followed by blocking with 1% bovine serum albumin. The ELISA

was used to analyze vesicles after treatment with methanol and chloroform as

described in “Lipid analysis.” Puriﬁed GXM was used as a positive control. The

samples were incubated in the plate overnight at 4°C. The plates were then

washed ﬁve times with a solution of TBS supplemented with 0.1% Tween 20,

followed by incubation with MAb 18B7 for 1 h. The plate was again washed and

incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG1 for

1 h. Reactions were developed after the addition of p-nitrophenyl phosphate

disodium hexahydrate, followed by reading at 405 nm with a Multiscan MS

(Labsystem, Helsinki, Finland). The antibodies in this assay were all used at a

concentration of 1 ␮g/ml.

Extracellular formation of GXM-containing vesicles. To explore the possibil-

ity that cryptococcal vesicles were formed as a result of random aggregation of

lipids and GXM in the extracellular environment, minimal medium was supple-

mented with GXM (15 ␮g/ml) and inoculated with Cap67 cells, which are known

to produce vesicles (see Fig. 1, 3, and 5). After growth for 72 h at 30°C,

supernatants were collected and evaluated for GXM-containing vesicles. Alter-

natively, acapsular cells were grown in the absence of GXM and the polysaccha-

ride was added to culture supernatants at the same concentration after removal

of yeast cells, followed by incubation for1hat30°C. GXM-containing superna-

tants were centrifuged at 100,000 ϫ g, and the pellets were puriﬁed in Sepharose-

bound MAb 18B7. GXM in supernatants and 100,000 ϫ g pellets was then

quantiﬁed by capture ELISA. The negative control consisted of GXM determi-

nations in regular Cap67 cultures.

Capsule induction. Capsule expression in C. neoformans was induced as de-

scribed previously (42). Brieﬂy, C. neoformans was inoculated in Sabouraud

broth diluted 10 times in 50 mM MOPS (morpholinepropanesulfonic acid), pH

7.3. After incubation for 6 or 24 h at 37°C with shaking, the cells were recovered

by centrifugation for 10 min at 2,000 ϫ g and ﬁxed in 2% paraformaldehyde in

TBS. The ﬁxative agent was removed by washing the cells with PBS, and after

India ink staining, capsule expression was analyzed microscopically. Capsule

sizes, deﬁned as the distances between the cell wall and the outer border of the

capsule, were determined using ImageJ software, elaborated and provided by the

National Institutes of Health (NIH) (http://rsb.info.nih.gov/ij/). All experiments

were performed in triplicate sets and the results analyzed using Student’s t test.

Supernatant-associated vesicles produced as described above were then puriﬁed

by afﬁnity chromatography and different centrifugation steps as described pre-

viously in this section. The GXM concentrations in 100,000 ϫ g fractions under

the different conditions of capsule induction were normalized to the number of

cells in the culture after each condition of stimulation, as measured in a

Neubauer chamber.

GXM binding by acapsular cells. Acapsular C. neoformans cells (strain Cap67,

cells) were suspended in 100 ␮l of a puriﬁed vesicular suspension, with a

GXM concentration corresponding to 10 ␮g/ml. The suspension was incubated

for 12 h at 25°C and extensively washed with PBS, followed by ﬁxation with 4%

paraformaldehyde. The cells were further blocked for1hinPBS-bovine serum

albumin and incubated with MAb 18B7 (1 ␮g/ml) for1hatroom temperature,

followed by a ﬂuorescein isothiocyanate-labeled goat anti-mouse IgG (Fc-spe-

ciﬁc) antibody (Sigma). Yeast cells were ﬁnally observed with an Axioplan 2

(Zeiss, Germany) ﬂuorescence microscope. Images were acquired using a Color

View SX digital camera and processed with the software system analySIS (Soft

Image System). In control systems, MAb 18B7 was replaced by irrelevant anti-

bodies. In addition, vesicle preparations were simply removed from the experi-

mental system (negative control) or replaced by soluble, nonvesicular GXM

(positive control), puriﬁed as described elsewhere (6).

Production of GXM-containing vesicles during the in vitro infection of mac-

rophages with C. neoformans. The ability of cryptococci to produce GXM-con-

taining vesicles during the infection of host cells was evaluated using the J774.16

cell line, which has been extensively used to study C. neoformans-mouse macro-

phage interactions. Macrophage-like cells were cultured in Dulbecco’s modiﬁed

Eagle’s medium, supplemented with 10% fetal calf serum, 10% NCTC109 cells,

and 1% nonessential amino acids, at 37°C in a 5% CO

atmosphere. Cultures

were also supplemented with 100 U/ml gamma interferon and 1 ␮g/ml lipopoly-

saccharide. The cells were grown to conﬂuence in 75-cm

ﬂasks and then infected

with C. neoformans (10 yeast cells per host cell) in the presence of 10 ␮g/ml of

MAb 18B7, which binds to the capsular polysaccharide and is opsonic (3). After

1 h of incubation at 37°C, free C. neoformans cells were removed by washing

them. Based on the observation that J774.16 cells lyse after hosting intracellular

replication of cryptococci (38), washed infected cells were incubated for 18 h at

37°C. Host cell lysis and cryptococcal intracellular replication were observed

microscopically. Fluids from infected cultures were then analyzed for vesicles as

described above.

RESULTS

Production of extracellular vesicles by C. neoformans. Sev-

eral lines of evidence suggested us that secretion of macromol-

ecules by fungi could rely on vesicular transport (11, 14, 30–32,

35, 36, 40), and consequently, we designed experiments to

search for vesicles in fungal cells and culture supernatants. In

acapsular cells, putative vesicular bodies were observed in as-

sociation with the cell wall (Fig. 1A to C), providing additional

evidence for the existence of an extracellular vesicular trans-

port mechanism. To address whether vesicles could be ob-

served during infection by encapsulated cryptococci, the pres-

ence of vesicles in vivo was evaluated by electron microscopy of

C57BL/6 mice infected intratracheally with C. neoformans.

TEM analysis demonstrated extracellular vesicular structures

near the edge of the capsule 2 h after infection (Fig. 1D) as

well as hypolucent vesicular structures in the cryptococcal cell

wall (Fig. 1E). Similar results were observed when mice were

killed 48 h or 7 days after infection (data not shown).

These results implied that cryptococcal vesicles could pass

through the cell wall to be released to the extracellular envi-

ronment. Consequently, we hypothesized that these structures

would be found in culture supernatants. Transmission electron

microscopy of material recovered by concentration of C. neo-

formans culture supernatants followed by differential sedimen-

tation revealed the presence of vesicular bodies (Fig. 1F to H).

Vesicle size and electron density varied considerably, but es-

sentially, all of them corresponded to spherical bodies with

evident bilayered membranes.

GlcCer and sterols are present in vesicular lipid extracts.

The pellets obtained after centrifugation of culture superna-

tants at 100,000 ϫ g were extracted with different mixtures of

organic solvents, and these preparations were analyzed by

50 RODRIGUES ET AL. EUKARYOT.CELL

HPTLC. In different vesicle preparations, bands with migra-

tion rates corresponding to the GlcCer standard were detected

in lipid extracts from both acapsular and encapsulated C. neo-

formans cells (Fig. 2A, inset). Vesicular extracts from both

encapsulated and acapsular isolates also present molecules

with R

values corresponding to that of ergosterol, the principal

fungal sterol. In order to conﬁrm that sterols and GlcCer are in

fact the molecules detected by HPTLC analysis, vesicle lipids

from encapsulated cells were examined by ESI (MS and MS/

MS) in positive mode. Although several molecular ions were

detected (Fig. 2), the analysis was focused on sterols and

GlcCer, the major molecules detected in HPTLC analysis.

The principal cerebroside produced by C. neoformans is

N-2Ј-hydroxyoctadecanoyl-1-␤-

D-glucopyranosyl-9-methyl-4,8-

sphingadienine, as demonstrated by our group (30). The full

spectra (scanned from 300 to 800 m/z) of neutral lipids ob-

tained after Folch’s partition in fact suggested the presence of

GlcCer with Li

adducts. Monolithiated peaks at m/z 734 ([M ϩ

]

) and 762 ([M ϩ Li

]

) were observed and revealed a

proﬁle similar to what has been described by our group and others

for fungal GlcCer (1, 24, 30). These molecular ions correspond to

N-2Ј-hydroxyhexadecanoyl- and N-2Ј-hydroxyoctadecanoyl-1-␤-

D-glucopyranosyl-9-methyl-4,8-sphingadienine, as conﬁrmed af-

ter ESI-MS/MS analysis (Fig. 2B and C). Loss of water ([M Ϫ

O ϩ Li

]

) generated peaks at m/z 716 and 744. Abundant

lithiated aglycon ([M Ϫ hexose ϩ Li

]

) peaks with m/z at 572

and 600 corresponded, respectively, to the loss of 162 units

from the molecular ions at m/z 734 and 762. The variation of 28

units among these peaks indicates the presence of two species

of hydroxylated fatty acids, containing 16 or 18 carbons. The

presence of an abundant ion peak at m/z 480 in both spectra,

corresponding to the loss of OH C

and OH C

fatty acids

([M Ϫ FA ϩ Li

]

), conﬁrmed this structural diversity.

Ergosterol and obtusifoliol are the major sterols produced

by C. neoformans (18). The major peaks at m/z 397 and 435

(Fig. 2) supported, respectively, the occurrence of a [M ϩ

]

ion corresponding to ergosterol and a [M ϩ Li

]

peak

compatible with a molecule differing from obtusifoliol by 2

FIG. 1. TEM of vesicles in acapsular (A to C) and encapsulated (D to H) C. neoformans cells. The occurrence of vesicles in association with

the cell wall of acapsular cryptococci (A and B) or in the extracellular environment (C) is evident after in vitro growth. Vesicle-like structures were

also observed in the lung following murine pulmonary infection (D and E). Putative vesicles near the edge of the capsule (D) or in the cryptococcal

cell wall (E) were observed 2 h after infection. Bars, 100 nm (A to C) and 500 nm (D and E). Arrows point to vesicles, and asterisks are on the

cryptococcal cell wall. (F to H) The pellets obtained by ultracentrifugation were isolated by differential centrifugation, puriﬁed from GXM by

afﬁnity chromatography, and analyzed by TEM. Extracellular vesicles with bilayered membranes and different proﬁles of electron density were

observed. Bars, 100 nm (F and G) and 50 nm (H).

OL. 6, 2007 GXM-CONTAINING VESICLES IN C. NEDFORMANS 51

units of mass. For structural elucidation, these ions were sub-

mitted to an ESI-MS/MS scan (Fig. 2D and E). The molecular

ion at m/z 397 ([M ϩ H]

) gave rise to fragments at m/z

285 ([C

), 302 ([C

]

), 190 ([C

]

), 173

([C

]

), and 154 ([C

]

), compatible with the sterol

proﬁle of fragmentation proposed by Scallen et al. (33) and

with a commercial standard of ergosterol (data not shown).

The fragmentation of the molecular ion at m/z 435 ([M ϩ Li]

)

yielded daughter ions at m/z 417 ([M Ϫ H

O ϩ Li

]

), 407

([C

OLi]

), 267 ([C

Li]

), and 186 ([C

Li]

Minor ions at m/z 391, 351, 337, 311, 295, 284, and 199 were

also observed. All of these peaks were compatible with a

monolithiated molecular ion corresponding to an obtusifoliol-

like molecule lacking the double bond between carbons 8 and

FIG. 2. Lipid analysis of C. neoformans vesicles. (A) Lipid extracts were prepared as described in Materials and Methods and analyzed by

ESI-MS and HPTLC. Vesicle lipids with migration rates corresponding to GlcCer and ergosterol standards (c) were detected by HPTLC (inset)

in preparations from both encapsulated (a) and acapsular (b) C. neoformans cells. ESI-MS analysis demonstrated the presence of molecular ions

corresponding to GlcCer (734 and 762) and sterols (435 and 397) besides other still-unidentiﬁed molecules (m/z values in gray). Fragmentation

analysis of molecular ions at m/z 734 (B) and 762 (C) revealed ionized species with m/z values typical of lithiated fungal cerebrosides. Ceramide

structures with C

(structure in panel B) and C

(structure in panel C) fatty acids were detected. Fragmentation analysis of molecular ions at m/z

397 (D) and 435 (E) revealed the presence of ergosterol (structure in panel D) and 4,14-dimethylergosta-24(24

)-en-3␤-ol (structure in panel E),

an obtusifoliol-like structure.

52 RODRIGUES ET AL. E

UKARYOT.CELL

9. This structure was therefore identiﬁed as 4,14-dimethyl-

ergosta-24(24

)-en-3␤-ol.

Vesicle production requires viable C. neoformans cells. After

growth of C. neoformans cells for 72 h, the viability of the cryp-

tococcal population was very close to 100%, as determined by

propidium iodide staining (data not shown). However, even with

high indices of viability, the possibility that the vesicles originated

from a minor fraction of dead cells could not be discarded. In this

context, we ﬁrst evaluated the ability of C. neoformans cells to use

radioactive precursors of ceramide to produce [

H]ceramide-con

taining vesicles, based on the evidence that GlcCer is present in

vesicle extracts. The detection of [

H]GlcCer in vesicle fractions

from strain ATCC 24067 (Fig. 3A) suggested that C. neoformans

cells metabolically incorporated the radioactive precursors and

secreted their corresponding products in vesicular preparations.

The insigniﬁcant levels of [

H]GlcCer detection in sterile media

containing the radioactive precursors and in 100,000 ϫ g super-

natants supported this hypothesis. In addition, GlcCer-containing

vesicles being transferred from the plasma membrane to the cell

wall were detected by immunogold labeling with antibodies to

GlcCer, followed by analysis by TEM (Fig. 3B). Wall-associated

extracellular vesicular bodies were also observed in these prepa-

rations.

The possibility that the vesicles could be the products of

dead cells was also considered, given that lipids are present in

the membranes of all organelles. Thus, we compared lipid

proﬁles obtained from regular vesicle fractions with those ob-

tained from dead cells. To avoid false-negative results, several

methods of fungal killing were evaluated, including metabolic

inhibition (sodium azide treatment) and protein denaturation

(mild heat exposure). C. neoformans cells were grown under

the conditions used for vesicle production, and then the super-

natant was collected and used for vesicle puriﬁcation. Cell

viability decreased by 83 or 99%, respectively, after treatment

with azide or heat (Fig. 3C). The treated yeast cells were

removed as described in Materials and Methods and the su-

pernatants centrifuged at 100,000 ϫ g. GlcCer was detected in

100,000 ϫ g fractions from living but not heat- or azide-treated

cells, as determined by HPTLC using a puriﬁed standard of

GlcCer. Sterol analysis revealed that compounds with migration

rates corresponding to an ergosterol standard were detected in

100,000 ϫ g fractions from supernatants of living and azide-

treated cells but not in preparations from heat-killed cryptococci.

We speculate that the detection of sterols in vesicles from azide-

treated cells could be the result of stress-associated secretory

activity during metabolic inhibition. Also, the sterol contents of

FIG. 3. Vesicle production requires cell viability. (A) The addition of radioactive ceramide precursors to the culture medium results in the

detection of signiﬁcant levels of radioactive GlcCer in vesicle preparations. In 100,000 ϫ g pellets from sterile medium and in 100,000 ϫ g

supernatants of grown cells, signiﬁcant levels of radioactive GlcCer were not detected (P Ͻ 0.001). (B) GlcCer-containing vesicles are apparently

transferred from the plasma membrane to the cell wall (a) and then secreted (b). Scale bars represent 100 nm. Arrows point to vesicles, and

asterisks are on the cryptococcal cell wall. (C) Killing of cryptococci with sodium azide or heating demonstrates a decrease in cell viability of 83%

(sodium azide) and 99% (heat), as determined by comparison with CFU counts obtained with untreated yeasts. Lipid analysis of 100,000 ϫ g

fractions of the supernatants obtained after fungal killing revealed the detection of GlcCer in fractions from living but not heat- or azide-treated

cells. Compounds with migration rates corresponding to an ergosterol standard were detected in 100,000 ϫ g fractions from supernatants of living

and azide-treated cells but not in preparations from heat-killed cryptococci. CPM, counts per minute.

VOL. 6, 2007 GXM-CONTAINING VESICLES IN C. NEDFORMANS 53

100,000 ϫ g fractions from living and azide-treated cells were

analyzed by densitometry, showing that the detection of sterol in

the former was 2.5-fold higher (data not shown). In summary,

these results suggest that the vesicles described here are produced

and excreted by living C. neoformans cells rather than released

from dead cells. The results obtained using the different strains

currently studied were very similar.

GXM is contained inside cryptococcal vesicles. The pres-

ence of GXM in vesicular bodies was evaluated by immuno-

gold labeling of puriﬁed vesicles with MAb 18B7, followed by

TEM analysis (Fig. 4A). Antibody binding to puriﬁed vesicles

was concentrated largely in the vesicular matrix. The presence

of GXM was conﬁrmed by the fact that the antibody reacted

strongly with the vesicles’ contents (obtained by solvent-medi-

ated lysis) in ELISAs (Fig. 4B). As an additional control, the

supernatant obtained before vesicle lysis was also assayed, and

the content of GXM in this preparation was around 10-fold

lower than the content of GXM in the vesicle pellet. The most

straightforward interpretation of these results is that GXM is

contained primarily in the intravesicular compartment but that

some material is found in solution, probably as a result of

vesicle damage and/or disruption in handling. To conﬁrm the

premise that GXM was intravesicular, we removed the extra-

cellular GXM by sequential passage of the vesicle preparation

over a column of Sepharose-bound MAb 18B7 (Fig. 4C). In

this experiment, the vesicle fraction and a vesicle-depleted

supernatant were consecutively passed through the antibody-

containing resin. While the GXM content in the supernatant

seemed to decrease after each step of incubation with the

resin, it stabilized when the vesicle preparation was used.

Hence, we conclude that the vesicles contain GXM.

To exclude the possibility that GXM-containing vesicles

were formed extracellularly by random incorporation of the

polysaccharide into liposomes instead of secretion, the mini-

FIG. 4. GXM is present in puriﬁed vesicles. (A) Immunogold labeling of puriﬁed vesicles with MAb 18B7 revealed a preferential intravesicular

distribution of GXM, as observed in different vesicular bodies (a to c). A higher magniﬁcation of the boxed area shown in panel c demonstrates

the occurrence of bilayered membrane (d). Bars, 150 (a), 180 (b), 120 (c), and 30 (d) nm. (B) The presence of GXM inside the vesicles was

conﬁrmed by polysaccharide detection in puriﬁed 100,000 ϫ g fractions from culture supernatants (white bar). Supernatants obtained from these

suspensions were assayed as a control of vesicle integrity, and in fact, GXM was detected at low levels in these preparations (black bar), suggesting

that some of these structures may be disrupted. (C) GXM content of vesicle suspensions and vesicle-depleted supernatants after serial passage

through a column composed of Sepharose-bound MAb 18B7. Sequential passages of vesicle-depleted supernatant through this column result in

a rapid loss of reactivity, while the GXM content of the vesicular preparation is relatively constant. (D) GXM-containing vesicles are not formed

extracellularly, since the addition of the polysaccharide to culture supernatants during or after growth of Cap67 cells is not followed by its detection

in puriﬁed 100,000 ϫ g fractions.

54 RODRIGUES ET AL. E

UKARYOT.CELL

mal medium was supplemented with GXM and inoculated with

Cap67 cells. After 72 h of growth at 30°C, Cap67 supernatants

were collected for vesicle puriﬁcation. Alternatively, acapsular

cells were grown in the absence of GXM, and the polysaccha-

ride was added to culture supernatants after the removal of

yeast cells. Vesicle puriﬁcation in these systems followed by

polysaccharide determination demonstrated the absence of

signiﬁcant amounts of GXM in the 100,000 ϫ g fractions (Fig.

4D), suggesting that the association of GXM and vesicles was

not a simple artifact of polysaccharide incorporation into lipo-

somes or polysaccharide-facilitated formation of lipid vesicles.

Interestingly, no signiﬁcant loss of GXM in supernatants was

observed when the polysaccharide was added to the culture

after fungal growth. When the medium was supplemented with

GXM before inoculation of acapsular cells, however, only one-

third of the total amount of the polysaccharide initially added

was detected in supernatants. This result could be linked to the

well-known ability of Cap67 cells to incorporate GXM into

their cell walls. In fact, immunoﬂuorescence analysis with MAb

18B7 revealed that after growth of Cap67 cells in the presence

of GXM, their surface was stained by the antibody to polysac-

charide (data not shown).

GXM content in vesicles correlates with capsule synthesis.

If GXM were packaged inside vesicles for extracellular trans-

port, we hypothesized that the conditions that promoted

capsule growth would also be associated with an increase in

supernatant vesicles. To evaluate the possible correlation be-

tween capsule synthesis and vesicle secretion, cryptococci were

stimulated to produce capsule, and supernatants were evalu-

ated for vesicle content. Vesicles were obtained by ultracen-

trifugation at 100,000 ϫ g, and pellets were resuspended in

TBS for GXM analysis by capture ELISA. GXM concentra-

tions in 100,000 ϫ g fractions under the different conditions of

capsule induction were normalized to the number of cells in

the culture under each condition of stimulation. In parallel,

yeast cells were collected by centrifugation, washed in PBS,

and ﬁxed in 2% paraformaldehyde for microscopic observation

after India ink staining. After 24 h of incubation of cryptococci

in diluted Sabouraud broth, capsule expression was approxi-

mately twofold higher than in cells incubated for 6 h (Fig. 5A).

Similarly, the content of GXM in the vesicle fraction after 24 h

was twofold higher than that observed after incubation of cryp-

tococci for 6 h (Fig. 5B).

Incorporation of vesicular GXM into the surface of acapsu-

lar cryptococci. The correlation between capsule growth and

GXM content in the vesicle fraction suggested that C. neofor-

mans must have mechanisms to extract the polysaccharide

from vesicles. To evaluate this hypothesis, acapsular C. neofor-

FIG. 5. Association of capsule expression with supernatant accumulation of GXM-containing vesicles. (A) Capsule expression was higher in

yeast cells incubated for 24 h in capsule-inducing media. (B) The proﬁle of GXM detection in ultracentrifugation pellets was very similar to that

observed for determinations of capsule size (inset), suggesting a correlation between capsule expression and the production of vesicles containing

GXM. GXM concentrations in 100,000 ϫ g fractions under the different conditions of capsule induction were normalized to the number of cells

in the culture under each experimental condition.

OL. 6, 2007 GXM-CONTAINING VESICLES IN C. NEDFORMANS 55

mans cells were incubated in the presence of puriﬁed vesicles

and then evaluated by immunoﬂuorescence with MAb 18B7. A

strong ﬂuorescent reaction of the antibody with acapsular cells

was observed after incubation with the vesicle suspension (Fig.

6), suggesting that C. neoformans can release GXM from ve-

sicular bodies and uses it for capsule growth. Control yeasts,

which had not been incubated with MAb 18B7 or vesicle prep-

arations, presented very weak levels of ﬂuorescence. Yeast

cells incubated with nonvesicular puriﬁed GXM also became

encapsulated (data not shown), as extensively described in

previous studies.

Detection of GXM-containing vesicles from regular cultures

or infected macrophages after sucrose gradient separation.

Given the size diversity of the vesicles demonstrated in Fig. 1,

a more detailed analysis of vesicle production by C. neoformans

during its regular growth was performed using ultracentrifuga-

tion, followed by ﬂotation on a step sucrose gradient (15).

After ultracentrifugation for 18 h, 12 fractions were obtained.

HPLC analysis revealed the presence of peaks with retention

times similar to that of a standard GlcCer in all fractions (Fig.

7A). The area of each peak varied depending on the sucrose

concentration in each fraction. Analysis of the presence of

GXM in sucrose gradient fractions was performed using a

capture ELISA. The results shown in Fig. 7B indicate that the

FIG. 6. Acapsular cells of C. neoformans bind GXM from extracel-

lular vesicles. Cap67 cells were incubated in the presence of puriﬁed

vesicles and then analyzed by immunoﬂuorescence with MAb 18B7.

Control cells, which were not incubated with MAb 18B7, are shown in

the upper panels (a and b). Yeast cells that were incubated in the

presence of the vesicular preparation reacted strongly with the anti-

body to GXM, as shown in the lower panels (c and d). The left panels

(a and c) represent cryptococci analyzed under differential interfer-

ence contrast, while the right panels (b and d) show images in the

ﬂuorescence mode. Bar, 2 ␮m.

FIG. 7. Analysis of vesicle fractions obtained after ultracentrifugation and sucrose gradient separation. Fractions from supernatants of regular

cultures were extracted with chloroform-methanol mixtures and analyzed by HPLC. In each fraction, a single peak with a retention time (Rt)

corresponding to a GlcCer standard was detected. Analysis of the same fractions (solid line) or preparations obtained from infected macrophages

(dashed line) by capture ELISA revealed different proﬁles of GXM distribution, although the polysaccharide was always expressively detected in

the region of the gradient presenting the highest density. Lipid and polysaccharide analyses were performed at least three times, always presenting

similar proﬁles. O.D. 405 nm, optical density at 405 nm.

56 RODRIGUES ET AL. EUKARYOT.CELL

maximum indices of polysaccharide detection were measured

in the most concentrated sucrose fractions, suggesting that

packaging of GXM into lipid membranes results in highly

dense vesicles. The inﬂuence of experimental conditions on

GXM distribution in gradient fractions was analyzed by chang-

ing sucrose concentrations. Using different conditions, the

highest content of GXM was always found in the most con-

centrated sucrose fractions (data not shown).

Based on the property that macrophages (J774.16 cells)

infected with C. neoformans become lysed after prolonged

periods of incubation (38), supernatants of yeast-infected mac-

rophage cells were collected for vesicle puriﬁcation. After sep-

aration of these ﬂuids by ultracentrifugation associated with

sucrose gradient, the presence of GXM was analyzed in each

fraction. By comparison of the proﬁle observed in infection-

derived vesicles with that observed in regular cultures (Fig.

7B), two additional regions containing high levels of polysac-

charide were observed in preparations obtained from infected

macrophages. These results support the idea that C. neofor-

mans produces GXM-containing vesicles inside host cells or,

alternatively, induces the production of host-derived mem-

brane domains ﬁlled with polysaccharide, as previously sug-

gested (38).

DISCUSSION

The secretion of macromolecules (molecular weight, Ͼ1

million) by fungal cells is a puzzling topic. To reach the extra-

cellular environment, secreted molecules must cross the cell

wall molecular network, a porous but very rigid complex. In C.

neoformans, it was recently demonstrated that GXM is traf-

ﬁcked within cytoplasmic secretory vesicles (40), supporting a

model that capsular materials are synthesized in the Golgi and

targeted to the plasma membrane for exocytosis. However, it

remains unknown how the vesicles would reach the extracel-

lular environment to be used for capsule expression. In this

regard, we present evidence that the major virulence factor of

C. neoformans is secreted by a novel mechanism involving the

release of membrane vesicles through the cell wall. Vesicle

secretion is apparently not exclusively related to GXM trafﬁc,

since lipid-containing extracellular vesicles were observed in

acapsular cells.

The possibility that the cell wall of C. neoformans is perme-

able to the passage of intact vesicular structures was supported

by microscopic analysis of acapsular cells cultivated in vitro and

encapsulated cells from infected mice. Putative vesicular bod-

ies in association with the cell wall and in the extracellular

milieu were observed in both acapsular and encapsulated cells,

suggesting that C. neoformans can use vesicular transport to

secrete different compounds. Microscopic analysis of pellets

obtained after differential centrifugation of culture superna-

tants revealed intact vesicles ranging in size from 60 to 300 nm.

Despite this heterogeneity, the vesicles had the common ap-

pearance of being rounded and deﬁned by a lipid bilayered

membrane. Differences in electron density were observed, sug-

gesting heterogeneity in vesicular contents. The sizes of the

vesicles were consistent with those shown in Fig. 1 and with the

size predicted from the early electron microscopic studies that

showed intracellular GXM in what appeared to be vesicular

structures (11, 14, 40). In those studies, the vesicular structures

were 100 to 300 nm in diameter, which is within the size range

of the vesicles described here.

Glycosphingolipids, sterols, and glycosylphosphatidylinosi-

tol-anchored proteins form detergent-insoluble lipid rafts on

the plasma membrane (23). They are required for the process-

ing of surface proteins in yeasts, making part of the vesicles

that link the reticuloendothelial system to the Golgi to the

plasma membrane (34). In C. neoformans, it has been sug-

gested that GlcCer-containing vesicles migrate from the cell

membrane to the cell wall (1, 25, 30). In the present study, lipid

analysis by mass spectrometry revealed that a glycosphingo-

lipid (GlcCer) and sterols are components of extracellular ves-

icles, supporting the idea that they are enriched in lipid rafts.

The physiologic production of GlcCer as a vesicle component

was strengthened by the observation that [

H]glucosylceramide

was metabolically incorporated in vesicular fractions. Lipids

were not detected in supernatants from dead cells, strongly

suggesting that C. neoformans physiologically secretes vesicles.

The visualization of GlcCer-containing vesicle-like structures

in both cell wall and extracellular spaces of C. neoformans cells

supports the view that these structures are active products of

fungal cells. The relevance of these ﬁndings to the pathogen-

esis and control of cryptococcosis is supported by different

studies, which demonstrated that GlcCer induces the produc-

tion of antimicrobial antibodies during human infection (30)

and is the target of defensins from insect and plant cells (37).

More recently, it was demonstrated that GlcCer expression

regulates cryptococcal virulence and is essential for fungal

growth in neutral/alkaline pH in vitro and in vivo (28).

Sterol derivatives, including ergosterol and an obtusifoliol-

like molecule, were also characterized as lipid components of

vesicle membranes. Ergosterol and obtusifoliol have previously

been described as major sterol components of C. neoformans

(18), but to our knowledge, this is the ﬁrst demonstration of

4,14-dimethylergosta-24(24

)-en-3␤-ol in cryptococcal mem

branes. The presence of other classes of hydrophobic com-

pounds is evidently expected, and indeed, additional molecular

ions were observed in ESI-MS analysis. The lack of detection

of these molecules by HPTLC could be due to either small

amounts of each individual lipid component or nonappropri-

ated conditions of separation and staining. The fact that other

lipids are clearly relevant to the physiology and pathogenesis of

C. neoformans (16, 17, 21) justiﬁes the characterization of

other components of vesicle membranes.

GXM, the principal capsular polysaccharide of C. neofor-

mans, was detected in vesicle preparations by serological ap-

proaches. The possibility that polysaccharide aggregates were

contaminating 100,000 ϫ g fractions was discarded, since ves-

icle preparations were puriﬁed in a MAb 18B7-bound resin. A

deﬁnitive association between GXM and vesicles was obtained

by immunogold labeling of the vesicles with an anti-GXM

antibody followed by TEM analysis, which revealed that the

polysaccharide is indeed surrounded by extracellular mem-

brane domains. This information was supported by the facts

that vesicle formation was not an artifact of polysaccharide-

mediated effects on lipids (Fig. 4D) and that the polysaccha-

ride and GlcCer, a lipid marker of cryptococcal vesicles, were

concomitantly detected in fractions from a sucrose gradient. In

this analysis, the polysaccharide content is directly related to

VOL. 6, 2007 GXM-CONTAINING VESICLES IN C. NEDFORMANS 57

vesicular density, which is in agreement with the high viscosity

described for GXM (22).

McFadden and coworkers recently provided experimental

evidence suggesting that capsule growth involves the produc-

tion of GXM ﬁbers that are released from, rather than at-

tached to, the cell (22). In this model, capsule construction

would depend on the self-association of GXM ﬁbers, in which

newly synthesized molecules would be secreted into the extra-

cellular environment and further incorporated throughout the

capsule by becoming entangled in existing capsular material. A

new model of capsule growth was recently proposed whereby

the capsule grows by apical extension (41). Vesicular transport

to the capsular edge, followed by polysaccharide unloading and

polysaccharide self-assembly into a capsule, would provide a

potential mechanism for apical growth. Such a mechanism may

have an inherent error rate such that some vesicles could be

released to the extracellular space without being unloaded and

could accumulate in the culture supernatant. Alternatively,

material destined for capsule or extracellular secretion may be

found in different vesicular fractions such that the vesicles

recovered represent deliberate extracapsular polysaccharide

transport. We, therefore, hypothesized that C. neoformans

could release capsular polysaccharides inside vesicles, which

would be further lysed by still-unknown mechanisms. The fact

that the detection of GXM in 100,000 ϫ g fractions is higher

when capsule expression is more efﬁcient in C. neoformans

supports this idea. The observation that acapsular mutants can

bind GXM from puriﬁed vesicles indicates that C. neoformans

is indeed able to lyse vesicles and use its internal content,

possibly through the activity of exocellular lipases (5).

Our current results and previous reports (11, 14, 40) indicate

that vesicles are synthesized intracellularly and transferred to

the cell surface, from which they are secreted to the extracel-

lular environment. It remains unknown how vesicles with an

average diameter of 160 nm cross the fungal cell wall, a highly

complex and dynamic structure (25). One possible explanation

comes from the observation that the cell wall of Saccharomyces

cerevisiae presents regular pores of around 200 nm, which can

be increased to 400 nm under stress conditions (7). Therefore,

despite being a rigid and complex structure, the fungal cell wall

could allow the passive passage of particles in the range of 60

to 300 nm, dimensions that could accommodate the vesicles

described here. Another mechanism for extracellular transport

could involve motor proteins. In Aspergillus fumigatus, a 180-

kDa polypeptide recognized by antimyosin antibodies was

found to be concentrated in cell wall and plasma membrane

fractions of conidia (9). Immunoelectron microscopy with an

antimyosin antiserum revealed that the antigen is distributed

mainly under the plasma membrane region, although it is also

clearly detected in different layers of the cell wall. The char-

acterization of cell wall motor proteins in fungal cells will

possibly contribute to the understanding of how vesicles are

transported to the extracellular milieu. Our current conclu-

sions from electron microscopy analyses of acapsular and en-

capsulated C. neoformans cells point to the presence of vesicles

in different compartments of the cell surface, including the

capsule network of ATCC 24067 cells.

The immunological activity of GXM (for a review, see ref-

erence 39) and lipids (1, 24, 25, 30) has been demonstrated in

different studies. Consequently, the delivery of GXM in vesi-

cles to target host tissues could involve a twin payload of

immunomodulating molecules that can interfere with immune

function. Our current results and previous reports suggest that

C. neoformans secretes capsular components during the infec-

tion of different host cells. In macrophages infected by cryp-

tococci, GXM is released by ingested yeast cells, followed by

damage to the phagosomal membrane and cytoplasmic accu-

mulation of polysaccharide-containing vesicles (38). In the cur-

rent study, the distributions of GXM in fractions obtained

from ultracentrifugation associated with a sucrose gradient

markedly differed when culture ﬂuids of C. neoformans cells

and supernatants from infected macrophages were compared.

When macrophage-derived supernatants were analyzed, an ad-

ditional peak of polysaccharide detection was observed, which

could be explained by the production of vesicles of different

natures during the interaction of C. neoformans cells with these

macrophages, as previously suggested by Tucker and Casade-

vall (38). The currently presented data also indicate that C.

neoformans produces extracellular vesicles in vivo, supporting

the idea that vesicular transport is relevant for polysaccharide

release during infection. This result may have an impact on the

understanding of the immunomodulatory activity of GXM,

since its secretion during infection is accompanied by the

release of other immunologically active molecules, such as

GlcCer and other still-uncharacterized molecules.

In summary, we report the existence of membrane vesicles

containing capsular polysaccharide in C. neoformans cultures

and host cells infected with this pathogen. In contrast to pro-

karyotes, C. neoformans capsular polysaccharide is synthesized

in the cell body and transported extracellularly for capsule

assembly by a mechanism that involves the production of ves-

icles. Hence, our results and other reports (11, 14, 31, 32, 40)

suggest that the eukaryotic solution to the problem of capsular

assembly takes advantage of a sophisticated trans-cell wall

vesicular transport secretory mechanism that is not available in

prokaryotes. The discovery that polysaccharide is packaged in

vesicles containing immunologically active lipids has the po-

tential to revolutionize our views of capsule assembly, extra-

cellular polysaccharide shedding, and the mechanisms by which

GXM mediates immunosuppression.

ACKNOWLEDGMENTS

M.L.R. was the recipient of an International Fellowship for Latin

America and is supported by grants from Conselho Nacional de Des-

envolvimento Cientı´ﬁco e Tecnolo´gico (CNPq-Brazil), Fundac¸a˜o Uni-

versita´ria Jose´ Bonifa´cio (FUJB-Brazil), and Fundac¸a˜o de Amparo a

Pesquisa do Estado do Rio de Janeiro (FAPERJ-Brazil). K.M. is

supported by Programa de Nanocieˆncia e Nanotecnologia, MCT-

CNPq. L.N. is supported by CNPq-Brazil. A.C. is supported by NIH

grants AI033142, AI033774, AI052733, and HL059842.

We thank Eliandro Lima, Yvonne Kress, and the Albert Einstein

College of Medicine Analytical Imaging Facility staff for help with

electron microscopy. We also thank Igor C. Almeida and Sirlei Daffre

for support with mass spectrometry analysis. We are indebted to Jorge

Jose´ Bastos Ferreira for helpful discussions.

D.L.O. is a M.S. student at Instituto de Bioguimica Medica, UFRJ.

REFERENCES

1. Barreto-Bergter, E., M. R. Pinto, and M. L. Rodrigues. 2004. Structure and

biological functions of fungal cerebrosides. An. Acad. Bras. Cienc. 76:67–84.

2. Casadevall, A., J. Mukherjee, and M. D. Scharff. 1992. Monoclonal antibody

based ELISAs for cryptococcal polysaccharide. J. Immunol. Methods 154:

27–35.

3. Casadevall, A., W. Cleare, M. Feldmesser, A. Glatman-Freedman, D. L.

58 RODRIGUES ET AL. EUKARYOT.CELL

Goldman, T. R. Kozel, N. Lendvai, J. Mukherjee, L. A. Pirofski, J. Rivera,

A. L. Rosas, M. D. Scharff, P. Valadon, K. Westin, and Z. Zhong. 1998.

Characterization of a murine monoclonal antibody to Cryptococcus neofor-

mans polysaccharide that is a candidate for human therapeutic studies.

Antimicrob. Agents Chemother. 42:1437–1446.

4. Chang, Y. C., and K. J. Kwon-Chung. 1994. Complementation of a capsule-

deﬁcient mutation of Cryptococcus neoformans restores its virulence. Mol.

Cell. Biol. 14:4912–4919.

5. Chen, S. C., L. C. Wright, R. T. Santangelo, M. Muller, V. R. Moran, P. W.

Kuchel, and T. C. Sorrell. 1997. Identiﬁcation of extracellular phospholipase

B, lysophospholipase, and acyltransferase produced by Cryptococcus neofor-

mans. Infect. Immun. 65:405–411.

6. Cherniak, R., L. C. Morris, B. C. Anderson, and S. A. Meyer. 1991. Facili-

tated isolation, puriﬁcation, and analysis of glucuronoxylomannan of Cryp-

tococcus neoformans. Infect. Immun. 59:59–64.

7. de Souza Pereira, R., and J. Geibel. 1999. Direct observation of oxidative

stress on the cell wall of Saccharomyces cerevisiae strains with atomic force

microscopy. Mol. Cell. Biochem. 201:17–24.

8. Dromer, F., J. Salamero, A. Contrepois, C. Carbon, and P. Yeni. 1987.

Production, characterization, and antibody speciﬁcity of a mouse monoclo-

nal antibody reactive with Cryptococcus neoformans capsular polysaccharide.

Infect. Immun. 55:742–748.

9. Esnault, K., B. el Moudni, J.-P. Bouchara, D. Chabasse, and G. Tronchin.

1999. Association of a myosin immunoanalogue with cell envelopes of

Aspergillus fumigatus conidia and its participation in swelling and germina-

tion. Infect. Immun. 67:1238–1244.

10. Feldmesser, M., A. Casadevall, Y. Kress, G. Spira, and A. Orlofsky. 1997.

Eosinophil-Cryptococcus neoformans interactions in vivo and in vitro. Infect.

Immun. 65:1899–1907.

11. Feldmesser, M., Y. Kress, and A. Casadevall. 2001. Dynamic changes in the

morphology of Cryptococcus neoformans during murine pulmonary infection.

Microbiology 147:2355–2365.

12. Feldmesser, M., Y. Kress, P. Novikoff, and A. Casadevall. 2000. Cryptococcus

neoformans is a facultative intracellular pathogen in murine pulmonary in-

fection. Infect. Immun. 68:4225–4237.

13. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the

isolation and puriﬁcation of total lipids from animal tissues. J. Biol. Chem.

226:497–509.

14. Garcı´a-Rivera, J., Y. C. Chang, K. J. Kwon-Chung, and A. Casadevall. 2004.

Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular

trafﬁcking of capsular glucuronoxylomannan. Eukaryot. Cell 3:385–392.

15. Gutwein, P., S. Mechtersheimer, S. Riedle, A. Stoeck, D. Gast, S. Joumaa, H.

Zentgraf, M. Fogel, and D. P. Altevogt. 2003. ADAM10-mediated cleavage

of L1 adhesion molecule at the cell surface and in released membrane

vesicles. FASEB J. 17:292–294.

16. Heung, L. J., A. E. Kaiser, C. Luberto, and M. Del Poeta. 2005. The role and

mechanism of diacylglycerol-protein kinase C1 signaling in melanogenesis by

Cryptococcus neoformans. J. Biol. Chem. 280:28547–28555.

17. Heung, L. J., C. Luberto, A. Plowden, Y. A. Hannun, and M. Del Poeta. 2004.

The sphingolipid pathway regulates Pkc1 through the formation of diacyl-

glycerol in Cryptococcus neoformans. J. Biol. Chem. 279:21144–21153.

18. Ibrahim, A. S., H. Sanati, and M. A. Ghannoum. 1996. Lipids of Cryptococ-

cus neoformans, p. 156–167. In R. Prasad and M. A. Ghannoum (ed.), Lipids

of pathogenic fungi. CRC Press, Boca Raton, FL.

19. Kozel, T. R. 1995. Virulence factors of Cryptococcus neoformans. Trends

Microbiol. 3:295–299.

20. Larsen, R. A., P. G. Pappas, J. Perfect, J. A. Aberg, A. Casadevall, G. A.

Cloud, R. James, S. Filler, and W. E. Dismukes. 2005. Phase I evaluation of

the safety and pharmacokinetics of murine-derived anticryptococcal anti-

body 18B7 in subjects with treated cryptococcal meningitis. Antimicrob.

Agents Chemother. 49:952–958.

21. Mare, L., R. Iatta, M. T. Montagna, C. Luberto, and M. Del Poeta. 2005.

APP1 transcription is regulated by inositol-phosphorylceramide synthase

1-diacylglycerol pathway and is controlled by ATF2 transcription factor in

Cryptococcus neoformans. J. Biol. Chem. 280:36055–36064.

22. McFadden, D. C., M. De Jesus, and A. Casadevall. 2006. The physical

properties of the capsular polysaccharides from Cryptococcus neoformans

suggest features for capsule construction. J. Biol. Chem. 281:1868–1875.

23. Mun˜iz, M., and H. Riezman. 2000. Intracellular transport of GPI-anchored

proteins. EMBO J. 19:10–15.

24. Nimrichter, L., M. D. Cerqueira, E. A. Leita˜o, K. Miranda, E. S. Nakayasu,

S. R. Almeida, I. C. Almeida, C. S. Alviano, E. Barreto-Bergter, and M. L.

Rodrigues. 2005. Structure, cellular distribution, antigenicity, and biological

functions of Fonsecaea pedrosoi ceramide monohexosides. Infect. Immun.

73:7860–7868.

25. Nimrichter, L., M. L. Rodrigues, E. G. Rodrigues, and L. R. Travassos. 2005.

The multitude of targets for the immune system and drug therapy in the

fungal cell wall. Microbes Infect. 7:789–798.

26. Novikoff, A. B., and P. M. Novikoff. 1977. Cytochemical contributions to

differentiating GERL from the Golgi apparatus. Histochem. J. 9:525–551.

27. Oscarson, S., M. Alpe, P. Svahnberg, A. Nakouzi, and A. Casadevall. 2005.

Synthesis and immunological studies of glycoconjugates of Cryptococcus neo-

formans capsular glucuronoxylomannan oligosaccharide structures. Vaccine

23:3961–3972.

28. Rittershaus, P. C., T. B. Kechichian, J. C. Allegood, A. H. Merrill, Jr., M.

Hennig, C. Luberto, and M. Del Poeta. 2006. Glucosylceramide synthase is

an essential regulator of pathogenicity of Cryptococcus neoformans. J. Clin.

Investig. 116:1651–1659.

29. Rodrigues, M. L., C. S. Alviano, and L. R. Travassos. 1999. Pathogenicity of

Cryptococcus neoformans: virulence factors and immunological mechanisms.

Microbes Infect. 1:293–301.

30. Rodrigues, M. L., L. R. Travassos, K. R. Miranda, A. J. Franzen, S. Rozental,

W. de Souza, C. S. Alviano, and E. Barreto-Bergter. 2000. Human antibodies

against a puriﬁed glucosylceramide from Cryptococcus neoformans inhibit

cell budding and fungal growth. Infect. Immun. 68:7049–7060.

31. Sakaguchi, N. 1993. Ultrastructural study of hepatic granulomas induced by

Cryptococcus neoformans by quick-freezing and deep-etching method. Vir-

chows Arch. B 64:57–66.

32. Sakaguchi, N., T. Baba, M. Fukuzawa, and S. Ohno. 1993. Ultrastructural

study of Cryptococcus neoformans by quick-freezing and deep-etching

method. Mycopathologia 121:133–141.

33. Scallen, T. J., A. K. Dhar, and E. D. Loughran. 1971. Isolation and charac-

terization of C-4 methyl intermediates in cholesterol biosynthesis after treat-

ment of rat liver in vitro with cholestan-3 beta, 5 alpha,6 beta-triol. J. Biol.

Chem. 246:3168–3174.

34. Su¨tterlin, C., T. L. Doering, F. Schimmoller, S. Schroder, and H. Riezman.

1997. Speciﬁc requirements for the ER to Golgi transport of GPI-anchored

proteins in yeast. J. Cell Sci. 110:2703–2714.

35. Takeo, K., I. Uesaka, K. Uehira, and M. Nishiura. 1973. Fine structure of

Cryptococcus neoformans grown in vitro as observed by freeze-etching. J.

Bacteriol. 113:1442–1448.

36. Takeo, K., I. Uesaka, K. Uehira, and M. Nishiura. 1973. Fine structure of

Cryptococcus neoformans grown in vivo as observed by freeze-etching. J.

Bacteriol. 113:1449–1454.

37. Thevissen, K., D. C. Warnecke, I. E. Francois, M. Leipelt, E. Heinz, C. Ott,

U. Zahringer, B. P. Thomma, K. K. Ferket, and B. P. Cammue. 2004.

Defensins from insects and plants interact with fungal glucosylceramides.

J. Biol. Chem. 279:3900–3905.

38. Tucker, S. C., and A. Casadevall. 2002. Replication of Cryptococcus neofor-

mans in macrophages is accompanied by phagosomal permeabilization and

accumulation of vesicles containing polysaccharide in the cytoplasm. Proc.

Natl. Acad. Sci. USA 99:3165–3170.

39. Vecchiarelli, A. 2000. Immunoregulation by capsular components of Crypto-

coccus neoformans. Med. Mycol. 38:407–417.

40. Yoneda, A., and T. L. Doering. 2006. A eukaryotic capsular polysaccharide is

synthesized intracellularly and secreted via exocytosis. Mol. Biol. Cell 17:

5141–5152.

41. Zaragoza, O., A. Telzak, R. A. Bryan, E. Dadachova, and A. Casadevall.

2006. The polysaccharide capsule of the pathogenic fungus Cryptococcus

neoformans enlarges by distal growth and is rearranged during budding. Mol.

Microbiol. 59:67–83.

42. Zaragoza, O., and A. Casadevall. 2004. Experimental modulation of capsule

size in Cryptococcus neoformans. Biol. Proced. Online 6:10–15.

VOL. 6, 2007 GXM-CONTAINING VESICLES IN C. NEDFORMANS 59

Anexo 2:

This article appeared in a journal published by Elsevier. The attached

copy is furnished to the author for internal non-commercial research

and education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling or

licensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of the

article (e.g. in Word or Tex form) to their personal website or

institutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies are

encouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation

reveals an intimate association between membrane lipids

and glucuronoxylomannan

Débora L. Oliveira

, Leonardo Nimrichter

, Kildare Miranda

, Susana Frases

c,d,1

, Kym F. Faull

Arturo Casadevall

c,d,2

, Marcio L. Rodrigues

Laboratorio de Estudos Integrados em Bioquimica Microbiana, Instituto de Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro

21941590, Brazil

Laboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Bioﬁsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941590, Brazil

Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA

Departments of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA

Pasarow Mass Spectrometry Laboratory, Semel Institute for Neuroscience and Human Behavior and the Department of Psychiatry and Biobehavioral Sciences, David Geffen School

of Medicine, University of California, Los Angeles, CA 90095, USA

article info

Article history:

Received 3 August 2009

Accepted 2 September 2009

Available online 10 September 2009

Keywords:

Cryptococcus neoformans

GXM transport

Secretory vesicles

Cryoimmunoelectronmicroscopy

abstract

Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of

polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxyloman-

nan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported

to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthe-

sis and trafﬁc is extremely limited. In this paper we report the GXM distribution in C. neoformans cells

sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and poly-

saccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular

organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that

cytoplasmic structures associated to vesicular compartments and reticular membranes are in close prox-

imity to the polysaccharide. GXM was generally found in association with the membrane of intracellular

compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryp-

tococcal supernatants by transmission electron microscopy in combination with serologic, chromato-

graphic and spectroscopic methods revealed fractions containing GXM and lipids. These results

indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consis-

tent with polysaccharide synthesis and transport in membrane-associated structures.

1. Introduction

The most prominent feature of the fungal pathogen Cryptococ-

cus neoformans is the presence of a capsule surrounding the yeast

cell body. This structure is composed mainly of polysaccharides,

including glucuronoxylomannan (GXM) and galactoxylomannan

(GalXM) (Zaragoza et al., 2009). The cryptococcal capsule is crucial

for fungal cell survival in different situations, including environ-

mental interactions with predators (Steenbergen et al., 2001)or

evasion of the immune system during human and animal infec-

tions (Monari et al., 2008; Vecchiarelli, 2007). GXM is considered

the most important virulence factor of C. neoformans (McClelland

et al., 2005) and, for this reason, it is also the most well studied

structural component.

GXM biosynthesis involves several enzymatic steps, starting

with sugar polymerization using nucleotide-activated sugar mono-

mers as precursors (Wills et al., 2001; Zaragoza et al., 2009). In con-

trast to cell wall polysaccharides like chitin and glucans, which are

most probably synthesized at plasma membrane level, GXM epi-

topes are found at intracellular sites (Feldmesser et al., 2001;

Garcia-Rivera et al., 2004; Yoneda and Doering, 2006), that pre-

sumably reﬂect the sites of initial assembly. Mutation in the gene

coding for a Rab/GTPase ortologue of the Saccharomyces cerevisiae

Sec4p caused accumulation of cytoplasmic GXM-containing vesi-

cles (Yoneda and Doering, 2006), raising the possibility that newly

synthesized polysaccharides might be exported to the cell surface

by vesicular trafﬁc. Since Sec4p is involved in post-Golgi secretion

doi:10.1016/j.fgb.2009.09.001

* Corresponding author.

E-mail addresses: [email protected], [email protected] (M.L.

Rodrigues).

Pressent address: Instituto Nacional de Metrologia, Normalizacao e Qualidade

Industrial, 50 Ave. NS das Gracas, Xerem 25259-020 Duque de Caxias, RJ, Brazil.

These authors share senior authorship.

Fungal Genetics and Biology 46 (2009) 956–963

Contents lists available at ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier.com/locate/yfgbi

Author's personal copy

events, it was deduced that GXM was at least partially synthesized

at the Golgi apparatus (Yoneda and Doering, 2006). GXM-contain-

ing vesicles are released to the extracellular milieu (Rodrigues

et al., 2008, 2007) in a process that apparently involves the SEC6

gene (Panepinto et al., 2009). Little is known about the biogenesis

of extracellular GXM-containing vesicles. Although different stud-

ies suggest a role of genes related to conventional post-Golgi secre-

tion in vesicle release (Panepinto et al., 2009; Yoneda and Doering,

2006), proteomic analysis of isolated C. neoformans vesicles reveled

similarities with mammalian exosomes (Rodrigues et al., 2008).

Our knowledge on the steps of GXM biosynthesis and cellular

trafﬁc is limited by the paucity of information on its intracellular

distribution. Previous ultrastructural analysis of C. neoformans cells

by immunoelectron microscopy (IEM) revealed that antibodies

raised against GXM recognize intracellular sites (Feldmesser

et al., 2001; Garcia-Rivera et al., 2004; Yoneda and Doering,

2006, 2009). Conventional IEM procedures, however, include sev-

eral steps of dehydration and embedding with different resins,

which markedly affects preservation of the cell envelope and intra-

cellular structures and organelles, and generates structural arti-

facts. Such subcellular modiﬁcations usually include loss of

cytoplasmic content, poor preservation of membranous structures

and cell wall shrinkage (Yamaguchi et al., 2005).

Cryoimmunogold labeling has been successfully used in differ-

ent studies of the C. neoformans biology (Rodrigues et al., 2008,

2007, 2000). Using this technique, in which cells are promptly

ﬁxed, frozen and sectionated, the artifacts that can plague conven-

tional IEM are reduced or avoided, and the result is superior pres-

ervation of subcellular structures, such as the nucleus, vacuoles

and mitochondria. In addition, cryosectioning has been suggested

to be the electron microscopy technique that is best suited for pre-

serving cellular epitopes (Slot and Geuze, 2007).

In a previous study by our group, we used a cryoultramicrotomy

protocol to study the cellular distribution of a glycolipid antigen in

C. neoformans (Rodrigues et al., 2000). Well preserved cells were

obtained, which allowed us to examine in detail different cellular

structures, including the cell wall and membranous structures. In

the present work, that method was used in association with vesicle

isolation and lipid analysis for the study of the distribution of GXM

in C. neoformans. Our data revealed that the polysaccharide is asso-

ciated with lipid matrices and previously unknown subcellular and

extracellular structures, indicating that GXM trafﬁc in C. neofor-

mans involves still unclear cellular events that may include uncon-

ventional steps of secretion.

2. Material and methods

2.1. Fungal cells

C. neoformans strain H99 was grown in minimal medium com-

posed of dextrose (15 mM), MgSO

(10 mM), KH

(29.4 mM),

glycine (13 mM), and thiamine-HCl (3

M). Fungal cells were cul-

tivated with shaking in Erlenmeyer ﬂasks at 30 °C for 72 h. Cells

were collected by centrifugation and then processed for IEM.

2.2. Electron microscopy

Electron microscopy procedures were based on the studies by

Tokuyasu (1973) and Rodrigues and colleagues (2000)). The cells

were ﬁxed in 0.1 M sodium cacodylate buffer (pH 7.2) containing

4% paraformaldehyde, 0.2% glutaraldehyde, and 1% picric acid for

60 min at room temperature. The cells were then washed three

times in PBS, inﬁltrated in gelatin, cut into cubes of 1 mm and inﬁl-

trated in 25% polyvinylpyrrolidone (PVP) and 2.3 M sucrose over-

night in a cold room. The polyvinylpyrrolidone embedded blocks

were then mounted on cryoultramicrotome stubs and ﬂash frozen

by immersion in liquid nitrogen. After trimming, ultrathin cryo-

sections were obtained at a temperature of À130 °C using an

Ultracut UCT cryoultramicrotome (Reichert). Flat ribbons of sec-

tions were shifted from the knife edge with an eyelash and picked

up in a wire loop ﬁlled with a drop of 1% (w/v) methyl cellulose,

2.3 M sucrose in PBS buffer. Sections were thawed on the pickup

droplet and transferred, sections downwards, to Formvar carbon-

coated nickel grids.

For immunolabelling, grids were passed over a series of droplets

of washing and were blocked in PBS-bovine serum albumin 1%.

After blocking the cryosections were incubated overnight in the

presence of 1

g/ml of the murine monoclonal antibody (mAb) to

GXM 18B7, washed again and incubated for 1 h with 15 nm (parti-

cle size) gold-labeled anti-mouse IgG. In control systems, the cryo-

sections were incubated with PBS instead of the mAb 18B7,

followed by the gold-labeled anti-mouse IgG. After a ﬁnal washing

series in PBS and distilled water, the sections were left for 15 min

on 2% uranyl acetate (aqueous) and transferred to 0.75% methyl

cellulose droplets. After 30 s, the grids were looped out, the excess

viscous solution was drained away and the sections were allowed

to dry. After drying, specimens were observed in a JEOL 1200EX

transmission electron microscope operating at 80 kV.

Transmission electron microscopy was used to analyze the

morphology and the integrity of extracellular vesicles isolated from

culture supernatants. Pellets obtained after centrifugation of cell-

free supernatants at 100,000g (see vesicle isolation and fractionation

below) were ﬁxed with 2% glutaraldehyde in 0.1 M cacodylate at

room temperature for 2 h, and then incubated overnight in 4%

formaldehyde, 1% glutaraldehyde, and 0.01 M PBS. The samples

were incubated for 90 min in 2% osmium, serially dehydrated in eth-

anol, and embedded in Spurrs epoxy resin. Thin sections were

obtained on a Reichart Ultracut UCT and stained with 0.5% uranyl

acetate and 0.5% lead citrate. Samples were observed in a JEOL

1200EX transmission electron microscope operating at 80 kV.

2.3. Vesicle isolation and fractionation

Isolation of extracellular vesicles was based on a protocol de-

scribed by Rodrigues et al. (2007). Cell-free culture supernatants

were obtained by sequential centrifugation at 5000 and 15,000g

(15 min, 4 °C) for removal of cells and debris. These supernatants

were concentrated approximately 20-fold using an Amicon ultraﬁl-

tration system (cutoff 100 kDa). The concentrate was again

sequentially centrifuged at 4000 and 15,000g (15 min, 4 °C) and

the remaining supernatant was then ultracentrifuged at 100,000g

for 1 h at 4 °C. The supernatant was discarded, and the pellet was

washed by ﬁve sequential suspension and centrifugation steps,

each consisting of 100,000g for 1 h at 4 °C with 0.1 M Tris-buffered

saline. To remove extravesicular GXM contamination, vesicles

were subjected to passage through a column packed with cyano-

gen bromide-activated Sepharose coupled to a monoclonal anti-

body to GXM, as described previously (Rodrigues et al., 2007).

Ultracentrifugation pellets were ﬁnally fractionated in Optiprep

gradients, as previously described for the analysis of virions and

mammalian exosomes (Cantin et al., 2008). Optiprep gradients

were prepared in PBS as 11 steps of 300

l in 1.2% increments

ranging from 6% to 18%. Vesicles were layered onto the top of

the gradient and ultracentrifuged at 250,000g for 75 min. Eleven

fractions of the gradient were collected from top to bottom. Each

fraction was then analyzed for the presence of sterols and GXM.

2.4. Lipid analysis

The pellets obtained from centrifugation of cell supernatants at

100,000g were ﬁrst suspended in methanol, and then two volumes

D.L. Oliveira et al. / Fungal Genetics and Biology 46 (2009) 956–963

957

Author's personal copy

of chloroform were added. The mixture was vigorously vortexed

and centrifuged to separate immediately formed polysaccharide-

containing precipitates. The supernatant was removed, dried with

a nitrogen stream, and partitioned according to Folch et al. (1957).

The lower phase, containing neutral lipids, was recovered and

dried by vacuum centrifugation. The dry residue was then pre-

pared for phospholipid analysis by combined liquid chromatogra-

phy–tandem mass spectrometry (LC/MS/MS) or sterol analysis by

thin-layer chromatography (TLC). For sterol analysis, the lipid ex-

tract was loaded into TLC silica plates (Si 60F254s; LiChrospher,

Germany) and separated using a solvent system containing

hexane:ether:acetic acid (80:40:2, vol:vol:vol) solvent. The plate

was sprayed with a solution of 50 mg ferric chloride (FeCl

)ina

mixture of 90 ml water, 5 ml acetic acid, and 5 ml sulfuric acid.

Sterol spots were identiﬁed by the appearance of a purple color

after heating the plates at 100 °C for 3–5 min. For phospholipid

identiﬁcation, the dry residue was suspended in 90% methanol.

After centrifugation, aliquots of the supernatant (typically 100

were injected onto a reverse phase HPLC column (Supleco

Ascentis

Express, C18, 150 Â 2.1 mm) equilibrated in buffer A

(methanol/water, 95/5, v/v, containing 1 mM ammonium acetate),

and eluted (100

l/m) with an increasing concentration of buffer B

(chloroform/water, 500/0.2, v/v, containing 1 mM ammonium ace-

tate; min/%B, 0/0, 5/0, 55/100). The efﬂuent from the column was

split, with a portion (about 70%) directed to a fraction collector

(1 min fractions) and the remainder (about 30%) passed to an Ion-

spray

source connected to a triple quadrupole mass spectrometer

(PE Sciex API III

) operating in the precursor (parent) scan mode in

which Q1 was scanned (0.3 Da step, 6.7 s/scan, oriﬁce 50 V), the

collision chamber was ﬂooded with argon gas (collision gas thick-

ness setting at 120), and Q3 was set to transmit ions at m/z

184.1 Da.

To conﬁrm assignments made from the LC/MS/MS data, and to

further deﬁne the composition of the phosphocholine lipids in

the sample, positive and negative ion mass spectra were recorded

off-line by direct injection of aliquots of fractions collected during

LC/MS/MS. Fractions 24–29 were pooled, and aliquots (20

l) were

injected into a stream of methanol/chloroform (4/1, v/v; 50

l/m)

entering the Ionspray

source of the mass spectrometer, before

and following the addition of formic acid (1

l, positive ion mode)

and triethylamine (1

l, negative ion mode). These experiments

were done with the mass spectrometer operating in either the nor-

mal (Q1 scan range m/z 200–1500, oriﬁce 70 V, 4.6 s/scan), or the

tandem mass spectrometric modes (precursor and fragment scan

modes). In particular, negative ion fragment scans at high oriﬁce

voltage (120) and relatively high collision gas density (CGT setting

150), were used to assign phosphocholine fatty acid substituents

after the addition of triethylamine. These experiments were done

using methods originally described by Jensen et al. (1986). Putative

parent ions for these experiments were assigned from the results

of the LC/MS/MS parent ion data, and conﬁrmed by the results

from the direct injection analyses. The parent ions were assigned

as 16 Da lighter than that determined experimentally, with the loss

of 15 Da predicted to result from source fragmentation at high ori-

ﬁce voltage (loss of one nitrogen methyl group), and 1 Da loss

resulting from abstraction of the proton attached to the phospho-

rous oxygen under basic conditions. Data was analyzed with

instrument-supplied software (MacSpec version 3.3), and optimal

conditions for the experiments were established using authentic

di-palmitoylphosphatidyl choline, heptadecanoyl-lyso-phosphati-

dyl choline and di-heptanoylphosphatidyl choline standards.

2.5. GXM capture ELISA

A 96-well polystyrene plate was coated with the capture IgM

mAb 2D10 (10

g/ml) for 1 h and then blocked with 1% bovine ser-

um albumin for. The plates were then incubated with GXM frac-

tions from vesicle samples, obtained by precipitation with a

mixture of chloroform and methanol (see item lipid analysis). After

three washing cycles with TBS 0,05% Tween 20, the plates were

incubated with an IgG1 to GXM (mAb 18B7, 2

g/ml) for 1 h at

room temperature. Positive reactions were developed by incuba-

tion with an alkaline phosphatase-conjugated anti-IgG1 followed

by reaction with p-nitrophenyl phosphate disodium hexahydrate

for 30 min. Absorbance values were measured at 405 nm in a

microplate reader.

3. Results and discussion

Electron microscopy procedures for the study of the C. neofor-

mans biology are complex because this fungal species is sur-

rounded by a polysaccharide capsule, which is attached to a thick

cell wall. The combination of a large capsule and thick cell wall

can slow ﬁxation process resulting in cellular degradation. Hence,

obtaining high quality micrographs of cryptococcal cells can take

some trial and error to identify the best conditions for ﬁxation.

In this context, the commonly used protocols for immunogold

analysis of C. neoformans usually include particular steps, such as

partial digestion of the cell wall (Yoneda and Doering, 2006), to en-

sure a more efﬁcient process of embedding and consequent preser-

vation of intracellular compartments. Unfortunately, these

procedures are often accompanied by loss of surface components

and, consequently, produce micrographs that convey limited infor-

mation on the cryptococcal cellular anatomy (Yamaguchi et al.,

2005). The advantages of using cryosectioning techniques have al-

ready been demonstrated in the model yeast S. cerevisiae, where

protocols based on the method of Tokuyasu (1973) resulted in high

resolution images with well preserved cellular structures and efﬁ-

cient immunogold labeling (Grifﬁth et al., 2008). The general pro-

cedures to prepare biological samples according to this method

include ﬁxation, inﬁltration/cryoprotection and rapid freezing in li-

quid nitrogen.

In the current study cryoultramicrotomy allowed the genera-

tion of well-preserved C. neoformans sections (Fig. 1). Intracellular

compartments such as the nucleus, vacuoles, mitochondria, cell

wall and capsule were clearly distinguishable. Even in the absence

of a post-ﬁxation step with OsO

, lipid bilayers were well pre-

served and easily discernible, as evidenced in nuclear compart-

ments and mitochondrial lamellas (Fig. 1). In addition to the

better known organelles, other subcellular structures were evident,

including reticular membrane clusters close to the plasma mem-

brane (Fig. 1C, black arrows). These compartments showed mor-

phological similarities to structures derived from the

endoplasmic reticulum, although their peripheral position inside

the cell suggested early endosomes (Gould and Lippincott-Sch-

wartz, 2009). A membranous compartment that resembled the

endoplasmic reticulum was also visible in the proximity of nucleus

(Fig. 1C). Finally, vesicular structures manifesting electron dense

contents were also apparent (Fig. 1B). These structures had similar-

ities to intermediate/late endosomal compartments called acido-

somes (Allen et al., 1993) and also to mammalian melanosomes

at different maturation stages (Raposo and Marks, 2007). These

compartments, which were consistently observed in our prepara-

tions, are shown in more detail in Fig. 2. Similar intracellular struc-

tures that contained GXM were recently described by Yoneda and

Doering using a secretion-deﬁcient mutant of C. neoformans

(Yoneda and Doering, 2009). However, the electron dense com-

partments observed in our model do not display such intense

GXM labeling (Fig. 2), as described in that study for C. neoformans

cells. In our model, some of the intracellular compartments also

contained internal vesicles (Fig. 1B and C) that show similarities

958 D.L. Oliveira et al. / Fungal Genetics and Biology 46 (2009) 956–963

Author's personal copy

to the so-called multivesicular bodies (MVBs) found in mammalian

cells (Gruenberg and Stenmark, 2004). The existence of MVBs in C.

neoformans was previously suggested using different techniques

(Rodrigues et al., 2008; Takeo et al., 1973), although differences

in morphology and electron density were noticed by comparing

the currently described results with those from previous studies.

Intracellular sites manifesting immunogold staining with mAb

to GXM were usually found in association with membranous struc-

tures (Fig. 3). Quantiﬁcation of immunogold labeling in intracellu-

lar compartments revealed that approximately 70% of the

antibody-binding sites were membrane-associated. On the basis

of this reactivity we tentatively assigned these areas as sites of

GXM synthesis/transport. Membrane compartments resembling

vesicles carrying GXM were repeatedly observed (Fig. 3), as well

as structures resembling mammalian MVBs (Fig. 3C) and periphe-

ral reticular clusters resembling endosomes (Fig. 3E). Since GXM

staining was often found in close association with the lipid bilayer,

we hypothesize that polysaccharide-binding proteins could be in-

serted in these lipid bilayers functioning as transient GXM anchors,

which still remains to be investigated. Considering that the size of

the complex gold-labeled antibody to GXM may in some instances

exceed the diameter of those structures, we cannot rule out the

possibility that they are located in neighboring intracellular re-

gions, although previous data indeed demonstrate that GXM asso-

ciated with secretory vesicles (Casadevall et al., 2009; Feldmesser

et al., 2001; Garcia-Rivera et al., 2004; Rodrigues et al., 2007;

Yoneda and Doering, 2006).

The intracellular, electron dense compartments described by

Yoneda and Doering (2009) were called sav1 bodies, since they

were most prominently observed in C. neoformans cells with

Fig. 1. Anatomy of the C. neoformans ultrastructure. C. neoformans cells were grown to stationary phase and prepared for EM as described in Section 2. Several organelles were

distinguished, including nucleus (N), vacuoles (V), mitochondria (M), cell wall (CW) and a compacted capsule (Cap). The existence of lamellar structures with unknown

cellular roles (black arrows) is suggested, as well as of vesicular electron dense compartments (white arrows). Black arrow heads indicate putative endoplasmic reticulum.

Unlabeled cells (A and B) and cells stained for the presence of GXM (C) are shown.

D.L. Oliveira et al. / Fungal Genetics and Biology 46 (2009) 956–963

959

Author's personal copy

Fig. 2. Electron dense organelles are present in C. neoformans. The boxed area shown in A is shown in higher magniﬁcation in B. Arrows indicate the electron dense bodies.

The composition of these electron dense structures is unknown. Cell surface labeling represents reactivity of cryptococcal components with mAb18B7.

Fig. 3. Cryptococcal GXM is found in association with vesicles and membranous structures. Cells were labeled with GXM binding mAb 18B7. GXM is generally detected in

association with lipid bilayers. In A and D, the arrowhead indicates that GXM can be localized inside vesicles that also present an electron dense content. In B and F, GXM

seems to be localized inside small cytoplasmic vesicle carriers. In F GXM is also presented inside the vacuole (V). In C, an unknown structure containing internal vesicles also

displays mAb18B7 labeling, as well as reticular unknown structures close to the plasma membrane displayed in E.

960 D.L. Oliveira et al. / Fungal Genetics and Biology 46 (2009) 956–963

Author's personal copy

defective expression of sav1p, a putative small GTPase involved in

post-Golgi secretion. The compartments observed in our model are

apparently distinct from the sav1 bodies, since they did not mani-

fest signiﬁcant reactivity with an antibody to GXM, despite mor-

phological similarities (Figs. 2 and 3A and D). The presence of

GXM in vacuoles (Fig. 3F) could be related to secretion mechanisms

initiated by endocytosis or to mechanisms of GXM recycling. In

fact, the GXM catabolic process is poorly understood and so far

hydrolytic enzymes able to cleave GXM have not been reported

in C. neoformans (Bose et al., 2003; Zaragoza et al., 2009).

Conventional procedures for TEM usually generate artiﬁcially

compacted cell wall structures, probably because of dehydration

steps. In our model, as well as in a previous study, cryoultramicr-

otomy procedures allowed the observation of structures that illus-

trate the thickness and complexity of the cell wall. As suggested in

previous studies with fungal cells (Soragni et al., 2001), clear dis-

tinctions between regions of different electron densities were ob-

served (Figs. 1B,C and 3F), which may be related to the

description of secondary cell walls during stationary growth of C.

neoformans (Farkas et al., 2009). Most of the cellular sites recog-

nized by the mAb to GXM were found in association with the cell

wall or within the capsular region (Fig. 1C), corroborating with pre-

vious observations (Yoneda and Doering, 2006).

GXM has been proposed to be exported to the extracellular

space in secretory vesicles, suggesting an association of lipids

and polysaccharides in C. neoformans (Rodrigues et al., 2007). This

notion is supported by our current observations in the cryptococcal

cytoplasm. Glucosylceramide and sterols were previously

Fig. 4. Analysis of phosphatidylcholine (PC) in extracellular vesicles. (A) Transmission electron microscopy showing vesicle morphology in 100,000g fractions isolated from

culture supernatants. (B) Characterization of PC in vesicle fractions: (a) total ion current LCMSMS chromatogram obtained following injection of the crude vesicle preparation

onto a reverse phase column as described in Section 2. The mass spectrometer was operated in the MS/MS precursor (parent) scan mode in which the m/z value of parent ions

that give rise to the m/z 184.1 fragment are recorded, (b–d) electrospray ionization mass spectra of the three peaks shown in Figure A (Peaks 1–3, MS/MS positive ion

precursor (parent) scan mode, precursors of m/z 184.1) and (e) negative ion electrospray mass spectrum of the fragments derived from an m/z 766.7 parent ion under MS/MS

conditions after addition of triethylamine as described in Section 2. Calculated values: C

P 782.570 Da; C

P 784.586 Da; C

P 786.601 Da; C

279.232 Da.

D.L. Oliveira et al. / Fungal Genetics and Biology 46 (2009) 956–963

961

Author's personal copy

characterized as membrane components of the C. neoformans ves-

icles (Rodrigues et al., 2007), but other lipid components of GXM-

containing vesicles are still unknown. To evaluate the relationship

of lipids with GXM export, we ﬁrst tested whether extracellular

vesicle samples contained GXM by ELISA. Vesicles obtained from

encapsulated cells reacted with the antibody to GXM, while vesi-

cles from an acapsular mutant manifested a much lower reactivity

(data not shown). These results were in agreement with those de-

scribed by Rodrigues and co-workers (2007) and support the

hypothesis that the major capsular polysaccharide of C. neoformans

associates with lipids for extracellular export. Since phospholipids

are major membrane constituents in most cells, but also in extra-

cellular vesicles of the fungal pathogen Histoplasma capsulatum

(Albuquerque et al., 2008), we evaluated whether the crude frac-

tion of GXM-containing vesicles contained this class of molecules.

To ensure that lipid characterization did not include membrane

fractions from non-vesicular structures, the integrity and morphol-

ogy of 100,000g fractions isolated from culture supernatants was

analyzed by TEM. Vesicles in these fractions showed typical bilay-

ered membranes that generally formed round compartments in the

size range of 50–300 nm (Fig. 4A), which is in agreement with pre-

vious reports on the morphological characterization of fungal

extracellular vesicles (Rodrigues et al., 2008; Eisenman et al., in

press). These fractions were then used for phospholipid extraction.

Cryptococcal phospholipids include phosphatidylinositol, lyso-

phosphatidyl ethanolamine, cardiolipin, glycophospholipids, lyso-

phosphatidyl choline, phosphatidic acid, phosphatidyl

ethanolamine and phosphatidyl choline (PC) (Rawat et al., 1984).

PC was the major phospholipid component of C. neoformans cellu-

lar membranes (Rawat et al., 1984), so this molecule was selected

as a potential candidate component of cryptococcal vesicles. Sam-

ples were subjected to precursor ion scanning of the m/z 184.1

fragment. This ion corresponds to phosphorylcholine, and there-

fore represents a molecular marker for the presence of PC. Control

samples consisted of LCMS analysis of the solvent alone, which

showed no signiﬁcant peaks. From the vesicle samples, three

strong peaks were observed eluting between 24 and 29 min

(Fig. 4B, panel a). These peaks revealed parent masses of 782.7,

784.6 and 786.8 Da (Fig. 4B, panel b–d). LCMS analysis of two dif-

ferent samples produced essentially identical results (data not

shown). Fractions eluting between 24 and 29 min were pooled

and examined by direct ﬂow-injection MS and MS/MS. Fragmenta-

tion of the peak at m/z 782.7 generated a strong signal at m/z 184

(data not shown), conﬁrming the occurrence of PC. To analyze fatty

acid components, negative MS/MS analysis was performed after

addition of triethylamine. Analysis of the peaks at m/z 782.7 and

784.6 revealed the presence of related fragments at m/z 279.2

(Fig. 4B, panel e) and 281.0 (not shown), consistent with the pres-

ence of, respectively, fatty acids corresponding to C18:2 and C18:1.

The peak at m/z 786.8 did not yield a signiﬁcant response probably

because of limitations in amount of sample (data not shown).

In our study, intracellular GXM-containing vesicles were

repeatedly observed (Fig. 3), as well as several unlabeled vesicular

structures (Figs. 1C and 2). In this context, we evaluated whether

the distribution of GXM in extracellular vesicles could reﬂect the

results observed in cellular structures. Fractionation of vesicle

samples by gradient centrifugation followed by lipid and polysac-

charide

analysis

revealed a previously unknown compositional

proﬁle of extracellular fungal vesicles (Fig. 5). Sterol analysis was

used to track membrane-containing fractions, since ergosterol

has been characterized as an easily detectable component of vesic-

ular membranes (Rodrigues et al., 2007). Phospholipid analysis in

gradient fractions was not pursued due to poor sensitivity for lipid

detection in chromatographic methods.

Ergosterol was detected in all fractions obtained after ultracen-

trifugation. GXM-containing fractions of different densities

(fractions 4–6 and 10), as well as sterol-containing fractions that

showed virtually no reactivity with an antibody to GXM (fractions

1–2 and 7–9), were observed. In some cases, the amounts of

GXM and sterols in gradient fractions were directly correlated

Fig. 5. Fractionation of C. neoformans extracellular vesicles. A crude preparation of extracellular vesicles was fractionated in a 6–18% Optiprep velocity gradient. Eleven

fractions were recovered and individually tested for the presence of GXM by ELISA and ergosterol by thin-layer chromatography (TLC). TLC bands (sterol detection) were

quantiﬁed by densitometry. Sterol distribution in fractions (dotted line) and GXM content (bars) are shown.

962 D.L. Oliveira et al. / Fungal Genetics and Biology 46 (2009) 956–963

Author's personal copy

(e.g. fractions 4–6), but fractions with lower ergosterol content and

high GXM density were also observed (e.g. fraction 10). These re-

sults are consistent with previously noted heterogeneity in vesicle

size and composition (Albuquerque et al., 2008; Rodrigues et al.,

2008) and indicate that some vesicles are preferentially associated

with polysaccharide transport while others contain no GXM, as

noted in prior immunogold labeling studies (Rodrigues et al.,

2007). The observation of extracellular vesicle fractions with such

different properties indicate that the cellular events required for

vesicle biogenesis are multiple and complex, as previously sug-

gested by morphological analysis of cryptococcal extracellular ves-

icles (Rodrigues et al., 2008). Our results add to the evolving theme

an intimate and intriguing association between lipid and polysac-

charide components in C. neoformans capsule (Nicola et al., 2009;

Sebolai et al., 2007, 2008).

In this work we report the use of two methodological ap-

proaches for studying the process of polysaccharide synthesis

and transport: (1) a cryosectioning protocol for C. neoformans,

which allowed a distinguished resolution of the fungal cytoplasmic

content and surface structures, and (2) a technique for fractionat-

ing vesicles that separates them into GXM- and non-GXM contain-

ing groups. The cryosectioning EM method allowed us to discern in

more detail the distribution of GXM inside the cell. The vesicle

fractionation approach should allow ﬁner resolution studies of ves-

icle composition and content by biochemical and mass spectrome-

try analysis. The results of these studies revealed new details for

the mechanisms required for polysaccharide trafﬁc and raise new

questions about this complex cellular process. Of particular inter-

est was the association of GXM with membrane structures sug-

gesting that this polysaccharide is synthesized in close proximity

to lipids, possibly by anchored enzymes. The possibility of combin-

ing cryoultramicrotomy with other less invasive methods such as

high pressure freezing followed by freeze substitution and electron

tomography could potentially generate new information for the

study of still unknown cell biology attributes of cell-wall contain-

ing pathogens.

Acknowledgements

A.C. was supported by NIH Grants AI33774, HL59842, AI33142,

and AI52733. M.L.R., L.N. and K.M. were supported by Grants from

the Brazilian agencies CNPq and FAPERJ. We thank Leslie Gunther

and the Analytical Imaging Facility team at Albert Einstein College

of Medicine for their assistance with the electron microscopy

experiments. We also thank Jorge José Jó B. Ferreira for helpful dis-

cussions. DLO is a PhD student at Instituto de Bioquímica Médica,

UFRJ.

References

Albuquerque, P.C. et al., 2008. Vesicular transport in Histoplasma capsulatum:an

effective mechanism for trans-cell wall transfer of proteins and lipids in

ascomycetes. Cell Microbiol. 10, 1695–1710.

Allen, R.D. et al., 1993. Acidosomes: recipients of multiple sources of membrane and

cargo during development and maturation. J. Cell Sci. 106 (Pt 1), 411–422.

Bose, I. et al., 2003. A yeast under cover: the capsule of Cryptococcus neoformans.

Eukaryot. Cell 2, 655–663.

Cantin, R. et al., 2008. Discrimination between exosomes and HIV-1: puriﬁcation of

both vesicles from cell-free supernatants. J. Immunol. Methods 338, 21–30.

Casadevall, A. et al., 2009. Vesicular transport across the fungal cell wall. Trends

Microbiol. 17, 158–162.

Eisenman, H.C., Frases, S., Nicola, A.M., Rodrigues, M.L., Casadevall. A., Vesicle-

associated melanization in Cryptococcus neoformans. Microbiology, in press,

doi:10.1099/mic.0.032854-0.

Farkas, V. et al., 2009. Secondary cell wall formation in Cryptococcus neoformans as a

rescue mechanism against acid-induced autolysis. FEMS Yeast Res. 9, 311–320.

Feldmesser, M. et al., 2001. Dynamic changes in the morphology of Cryptococcus

neoformans during murine pulmonary infection. Microbiology 147, 2355–2365.

Folch, J. et al., 1957. A simple method for the isolation and puriﬁcation of total

lipides from animal tissues. J. Biol. Chem. 226, 497–509.

Garcia-Rivera, J. et al., 2004. Cryptococcus neoformans CAP59 (or Cap59p) is involved

in the extracellular trafﬁcking of capsular glucuronoxylomannan. Eukaryot. Cell

3, 385–392.

Gould, G.W., Lippincott-Schwartz, J., 2009. New roles for endosomes: from vesicular

carriers to multi-purpose platforms. Nat. Rev. Mol. Cell Biol. 10, 287–292.

Grifﬁth, J. et al., 2008. A cryosectioning procedure for the ultrastructural analysis

and the immunogold labelling of yeast Saccharomyces cerevisiae. Trafﬁc 9,

1060–1072.

Gruenberg, J., Stenmark, H., 2004. The biogenesis of multivesicular endosomes. Nat.

Rev. Mol. Cell Biol. 5, 317–323.

Jensen, N.J. et al., 1986. Fast atom bombardment and tandem mass spectrometry of

phosphatidylserine and phosphatidylcholine. Lipids 21, 580–588.

McClelland, E.E. et al., 2005. Coping with multiple virulence factors: which is most

important? PLoS Pathog. 1, e40.

Monari, C. et al., 2008. Capsular polysaccharide induction of apoptosis by intrinsic

and extrinsic mechanisms. Cell Microbiol. 10, 2129–2137.

Nicola, A.M. et al., 2009. Lipophilic dye staining of Cryptococcus neoformans

extracellular vesicles and capsule. Eukaryot. Cell. 8, 1373–1380.

Panepinto, J. et al., 2009. Sec6-dependent sorting of fungal extracellular exosomes

and laccase of Cryptococcus neoformans. Mol. Microbiol. 71, 1165–1176.

Raposo, G., Marks, M.S., 2007. Melanosomes – dark organelles enlighten endosomal

membrane transport. Nat. Rev. Mol. Cell Biol. 8, 786–797.

Rawat, D.S. et al., 1984. Lipid composition of Cryptococcus neoformans.

Microbiologica 7, 299–307.

Rodrigues, M.L. et al., 2008. Extracellular vesicles produced by Cryptococcus

neoformans contain protein components associated with virulence. Eukaryot

Cell. 7, 58–67.

Rodrigues, M.L. et al., 2007. Vesicular polysaccharide export in Cryptococcus

neoformans is a eukaryotic solution to the problem of fungal trans-cell wall

transport. Eukaryot. Cell. 6, 48–59.

Rodrigues, M.L. et al., 2000. Human antibodies against a puriﬁed glucosylceramide

from Cryptococcus neoformans inhibit cell budding and fungal growth. Infect

Immunol. 68, 7049–7060.

Sebolai, O.M. et al., 2007. 3-Hydroxy fatty acids found in capsules of Cryptococcus

neoformans. Can. J. Microbiol. 53, 809–812.

Sebolai, O.M. et al., 2008. The inﬂuence of acetylsalicylic acid on oxylipin migration

in Cryptococcus neoformans var. neoformans UOFS Y-1378. Can. J. Microbiol. 54,

91–96.

Slot, J.W., Geuze, H.J., 2007. Cryosectioning and immunolabeling. Nat. Protoc. 2,

2480–2491.

Soragni, E. et al., 2001. A nutrient-regulated, dual localization phospholipase A(2) in

the symbiotic fungus Tuber borchii. EMBO J. 20, 5079–5090.

Steenbergen, J.N. et al., 2001. Cryptococcus neoformans interactions with amoebae

suggest an explanation for its virulence and intracellular pathogenic strategy in

macrophages. Proc. Nat. Acad. Sci. USA 98, 15245–15250.

Takeo, K. et al., 1973. Fine structure of Cryptococcus neoformans grown in vitro as

observed by freeze-etching. J. Bacteriol. 113, 1442–1448.

Tokuyasu, K.T., 1973. A technique for ultracryotomy of cell suspensions and tissues.

Cell

Biol. 57, 551–565.

Vecchiarelli, A., 2007. Fungal capsular polysaccharide and T-cell suppression: the

hidden nature of poor immunogenicity. Crit. Rev. Immunol. 27, 547–557.

Wills, E.A. et al., 2001. Identiﬁcation and characterization of the Cryptococcus

neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact

on pathogenicity. Mol. Microbiol. 40, 610–620.

Yamaguchi, M. et al., 2005. Safe specimen preparation for electron microscopy of

pathogenic fungi by freeze-substitution after glutaraldehyde ﬁxation. Nippon

Ishinkin Gakkai Zasshi 46, 187–192.

Yoneda, A., Doering, T.L., 2006. A eukaryotic capsular polysaccharide is synthesized

intracellularly and secreted via exocytosis. Mol. Biol. Cell 17, 5131–5140.

Yoneda, A., Doering, T.L., 2009. An unusual organelle in Cryptococcus neoformans

links luminal pH and capsule biosynthesis. Fungal Genet. Biol. 46, 682–687.

Zaragoza, O. et al., 2009. The capsule of the fungal pathogen Cryptococcus

neoformans. Adv. Appl. Microbiol. 68, 133–216 (Chapter 4).

D.L. Oliveira et al. / Fungal Genetics and Biology 46 (2009) 956–963

963

Anexo 3:

INFECTION AND IMMUNITY, Apr. 2010, p. 1601–1609 Vol. 78, No. 4

0019-9567/10/$12.00 doi:10.1128/IAI.01171-09

Extracellular Vesicles from Cryptococcus neoformans

Modulate Macrophage Functions

ᰔ

†

De´bora L. Oliveira,

Ce´lio G. Freire-de-Lima,

Joshua D. Nosanchuk,

3,4

Arturo Casadevall,

3,4

Marcio L. Rodrigues,

‡ and Leonardo Nimrichter

‡*

Laborato´rio de Estudos Integrados em Bioquímica Microbiana, Instituto de Microbiologia Professor Paulo de Go´ es,

and

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,

Rio de Janeiro, Brazil; Department of

Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

; and Department of

Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

Received 16 October 2009/Returned for modiﬁcation 20 November 2009/Accepted 2 February 2010

Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the

cell wall and contain a varied assortment of components, some of which have been associated with virulence.

Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal

infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown.

In this report, we demonstrate by ﬂuorescence microscopy that mammalian macrophages can incorporate

extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages

resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-␣), interleukin-10 (IL-10), and

transforming growth factor ␤ (TGF-␤). Vesicle preparations also resulted in a dose-dependent stimulation of

nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce

antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results

indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently

activating these phagocytic cells to enhance their antimicrobial activity. These results establish that crypto-

coccal vesicles are biologically active.

Cryptococcus neoformans is an encapsulated yeast that

causes disease in diverse species, including humans. Infection

is most commonly acquired by inhalation of environmental

propagules. C. neoformans rarely causes disease in immuno-

competent individuals, but patients with immunological disor-

ders can develop disseminated and neural cryptococcosis (63).

Extracellular microbial products have been amply demon-

strated to modulate the interaction between host cells and

pathogens. Many virulence factors and immunogens are re-

leased in their soluble forms by fungal cells to the extracellular

space (4, 9, 16, 19, 37, 49, 53, 60, 62, 65, 67). C. neoformans, for

instance, constitutively secretes large amounts of its capsular

polysaccharide glucuronoxylomannan (GXM) (61). Disease

progress is associated with detection of GXM, which is a po-

tent modulator of the immune response (reviewed in reference

81). Other secreted virulence-related factors include galactoxy-

lomannan (GalXM) (14), phospholipases (16), and urease (12,

62). In addition to acting as virulence factors, culture super-

natant components are immunogenic, conferring protection

against C. neoformans infection (51, 53).

Phagocytes are particularly important effector cells in the

control of systemic mycoses (54). The interaction of C. neofor-

mans with phagocytes, including macrophages, monocytes,

dendritic cells, and neutrophils, has been widely studied (23,

32, 43, 46, 50, 59, 68, 77). Cryptococcal GXM is antiphagocytic

(34) and a powerful immunomodulator (45, 79). C. neoformans

capsule size directly correlates with the efﬁcacy of phagocytosis

in vitro (6, 15, 82). Phagocytosis of C. neoformans can result in

either fungal killing (24, 30) or survival (2, 3, 39–41, 71, 80).

Killing of C. neoformans apparently involves the production of

oxidative species (24), while the mechanisms of fungal escape

include phagosome extrusion, cell-to-cell spread, and phago-

somal permeabilization (2, 3, 40, 41, 71). Capsular polysaccha-

rides and melanin are known to modulate the interaction of C.

neoformans with phagocytes in favor of the fungus (27, 39, 47,

48, 71, 72, 74, 76), but the role of other structures in the

outcome of yeast phagocytosis is virtually unknown.

A number of recent studies have shown that GXM, GalXM,

pigments, proteins, and lipids are trafﬁcked in vesicles that

traverse the cell wall (7, 14, 20, 56, 57, 62, 64, 65). Extracellular

vesicles are also produced by the pathogens Candida albicans,

C. parapsilosis, Sporothrix schenckii, and Histoplasma capsula-

tum, as well as by the model yeast Saccharomyces cerevisiae (1),

suggesting that extracellular vesicle secretion is a general prop-

erty of fungal cells. Secreted vesicles are heterogeneous. For

instance, vesicles secreted by C. neoformans were classiﬁed into

four different groups based on morphology and electron den-

sity (64). Additionally, vesicle diameter ranges from 30 to 400

nm, with the majority having dimensions of 100 to 150 nm (20,

64, 65). The combined use of serology, biochemistry, proteo-

mics, and lipidomics led to the identiﬁcation of 2 polysaccha-

rides, phospholipids, 4 neutral lipids, and 76 proteins as extra-

cellular vesicle components secreted by C. neoformans, which

* Corresponding author. Mailing address: Laborato´rio de Estudos

Integrados em Bioquímica Microbiana, Instituto de Microbiologia

Prof. Paulo de Go´es, Avenida Carlos Chagas Filho 373, Cidade Uni-

versita´ria, Centro de Cieˆncias da Sau´de, Bloco E, sub-solo, Rio de

Janeiro RJ 21941902, Brazil. Phone: 55 21 25626740. Fax: 55 21

25608344. E-mail: [email protected].

† Supplemental material for this article may be found at http://iai

.asm.org/.

‡ L.N. and M.L.R. share senior authorship on this article.

ᰔ

Published ahead of print on 9 February 2010.

1601

by on March 18, 2010 iai.asm.orgDownloaded from

means that at least 81 different molecules are released to the

extracellular milieu by vesicular secretion (14, 57, 64). It is

likely that this number is an underestimate resulting from the

difﬁculty of proteomic studies in vesicles from highly encapsu-

lated cryptococcal cells, since a higher number of vesicular

proteins were characterized in other fungi. For example, in H.

capsulatum, proteomics and lipidomics of extracellular vesicles

revealed an even more complex composition, including 283

proteins and 17 different phospholipids (1).

In this study, we evaluated the inﬂuence of extracellular

vesicles on the fate of C. neoformans after phagocytosis by

mouse macrophages. Our results show that fungal vesicles are

biologically active and stimulate macrophages. Moreover, our

results demonstrate that vesicles from an acapsular mutant

strain were more effective in eliciting macrophage activation

and augmenting fungal killing than vesicles from encapsulated

strains. Taken together, our ﬁndings suggest that fungal secre-

tory vesicles have the potential to inﬂuence the interaction of

C. neoformans with host cells.

MATERIALS AND METHODS

C. neoformans growth conditions. For vesicle isolation, C. neoformans strains

HEC3393 (serotype A, human isolate), B3501 (serotype D), and Cap 67 (sero-

type D acapsular strain, generated in a B3501 background [28]) were cultivated

for 72 h in a minimal medium composed of dextrose (15 mM), MgSO

(10 mM),

(29.4 mM), glycine (13 mM), and thiamine-HCl (3 M). Fungal cells

were cultivated at room temperature with continuous shaking. For macrophage

infection assays, fungal cells were cultivated in Sabouraud liquid medium for 48 h

at room temperature with continuous shaking. All media were prepared with

apyrogenic water, and glassware was rendered sterile and pyrogen free by heating

to 190°C for 4 h (18).

Macrophage cultures. The RAW 264.7 murine macrophage cell line (obtained

from the American Type Culture Collection [ATCC]) was cultivated in complete

Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal

calf serum (FCS), 2 mM

L-glutamine, 1 mM sodium pyruvate, 10 mg ml

Ϫ1

gentamicin, minimal essential medium (MEM) nonessential amino acids (cata-

log no. 11360; Gibco-Invitrogen), 10 mM HEPES, and 50 mM 2-beta-mercap-

toethanol. The cells were maintained at 37°C in a 7.5% CO

atmosphere. The

macrophage cultures and the experiments described below were all prepared

under lipopolysaccharide-free conditions.

Vesicle isolation. Isolation of extracellular vesicles was done using the protocol

described by Rodrigues et al. (65). After growth until the stationary phase (ϳ10

to 10

cells/ml), two liters of cell-free culture supernatants was obtained by

sequential centrifugation at 5,000 and 15,000 ϫ g (15 min at 4°C). The super-

natants were concentrated by approximately 20-fold using an Amicon ultraﬁl-

tration system (cutoff, 100 kDa). The concentrate was again sequentially centri-

fuged at 4,000 and 15,000 ϫ g (15 min at 4°C). The remaining supernatant was

then ultracentrifuged at 100,000 ϫ g for1hat4°C. The supernatant was

discarded, and the pellet was washed by ﬁve sequential suspension and centri-

fugation steps, each consisting of 100,000 ϫ g for1hat4°Cwith 0.1 M Tris-

buffered saline. To remove extravesicular GXM contamination, vesicles were

subjected to passage through a column packed with cyanogen bromide-activated

Sepharose coupled to a monoclonal antibody to GXM, as described previously

(65). Fractions that were not bound to the antibody-containing column were

again centrifuged at 100,000 ϫ g. The presence of extracellular vesicles in the

pellets produced by centrifugation at 100,000 ϫ g was checked by chemical

methods, as previously described (65). The quantiﬁcation of vesicle fractions was

performed based on the presence of sterols in their membranes by the use of a

quantitative ﬂuorimetric Amplex Red sterol assay kit (Molecular Probes). In

different assays, average values of 6 to 10 ␮g of sterol were detected as the ﬁnal

product of 2-liter cultures. The total sterol obtained after ultracentrifugation

suggests a recovery of 0.5 fg per yeast cell. The apparent low efﬁciency during

vesicle isolation was expected, since they could naturally disrupt in the superna-

tant. Vesicle suspensions were then adjusted to 30 to 50 ␮g of sterol/ml of

phosphate-buffered saline (PBS) for use in the subsequent experiments.

Visualization of incorporation of vesicular components by macrophages.

RAW 264.7 cells were plated onto the wells of a 24-well plate covered with glass

coverslips (5 ϫ 10

cells per well). Vesicle samples were stained with the li-

pophilic ﬂuorophore dialkylcarbocyanine iodide (DiI) (catalog no. V22885; In-

vitrogen) as described previously (55). Brieﬂy, vesicle suspensions were normal-

ized to a sterol concentration corresponding to 2 ␮g/ml and supplemented with

a DiI solution (ﬁnal concentration, 3 ␮M). After 30 min at room temperature,

the vesicle suspension was ultracentrifuged at 100,000 ϫ g for 1 h and washed

three times as described above. DiI-labeled vesicles were suspended in PBS to

form 2 ␮g/ml sterol solutions and then incubated with the macrophages for 30

min at 37°C in a 7.5% CO

atmosphere. Controls included culture medium

incubated with DiI alone and washed with PBS under the same conditions.

Vesicle-treated macrophages were then ﬁxed with 4% paraformaldehyde in PBS

for 5 min and blocked with PBS–5% bovine serum albumin (BSA) for1hat

room temperature. Plasma membranes and nuclei were labeled with cholera

toxin subunit B (CtxB) (1 ␮g/ml) and 4Ј,6-diamidino-2-phenylindole (DAPI) (10

␮g/ml), respectively, as described previously (75). Samples were then placed in

mounting medium (50% glycerol–50 mM N-propyl gallate–PBS) over glass slides

and visualized with a Leica AOBS laser scanning confocal microscope (Mann-

heim, Germany) with a 63ϫ oil immersion optic. z-axis series and single scan

images were obtained. The x- and z-axis sections were ﬁnally reconstructed using

ImageJ software (NIH [http://rsb.info.nih.gov/ij/]). Alternatively, cells incubated

with DiI-stained vesicles were sequentially blocked in PBS containing 5% BSA

and incubated for1hatroom temperature with pooled serum samples from 10

individuals diagnosed with cryptococcosis. As a negative control, cells were

incubated with normal human serum samples from healthy volunteers with no

previous diagnosis of any systemic mycosis. Pooled serum samples were used at

a 1:100 dilution in PBS–1% BSA. After washing, cells were incubated with Alexa

Fluor 488-labeled anti-human immunoglobulin antibody (Invitrogen). Samples

were then washed, placed in mounting medium as described above, and visual-

ized with an Axioplan 2 ﬂuorescence microscope (Zeiss, Germany). Images were

acquired using a Color View SX digital camera and processed with the analySIS

(Soft Image System) software system.

Cell viability. To evaluate whether the exposure of macrophage-like cells to

vesicle samples would lead to cell lysis, cell supernatants were assayed for the

presence of lactate dehydrogenase (LDH) activity. LDH is a cytoplasmic enzyme

retained by viable cells with intact plasma membranes. Membrane damage re-

sults in LDH release to culture supernatants. RAW 264.7 monolayers (1 ϫ 10

cells/well) were stimulated for 16 h with vesicle suspension (2 ␮g/ml sterol). After

this period, aliquots of the supernatant were collected and supplemented with

NADH and pyruvate to ﬁnal concentrations of 0.3 and 4.7 mM, respectively,

followed by 10 min of incubation at room temperature. Based on the property

that NADH, and not NAD, absorbs strongly at 340 nm, the decrease in absor-

bance (A

340

) was measured in a spectrophotometer. Positive and negative con-

trols consisted, respectively, of a RAW 264.7 Triton X-100 (10%) lysate and

supernatants of nontreated cells after 16 h of cultivation. All experiments were

performed in triplicate sets and statistically analyzed by Student’s t test.

Production of cytokines and nitric oxide (NO) after exposure of macrophages

to vesicle samples. RAW monolayers were washed twice in serum-free DMEM,

placed in medium supplemented with various concentrations of fungal vesicles,

and incubated for 16 h at 37°C (7.5% CO

atmosphere). In these experiments, we

used 40 ng of vesicular sterol to activate 10

macrophages, suggesting that 0.4 pg

of sterol is enough to activate a macrophage. As a positive control, macrophages

were stimulated with 1 ␮g/ml lipopolysaccharide (LPS). Supernatants were then

collected and assayed for interleukin-10 (IL-10), transforming growth factor ␤

(TGF-␤), and tumor necrosis factor alpha (TNF-␣) content by enzyme-linked

immunosorbent assay (ELISA) according to the manufacturer’s protocol (R&D

Systems). Alternatively, NO levels were measured in supernatants as previously

described (25). N6-(1-Iminoethyl)-

L-lysine dihydrochloride (L-NIL) (Cayman

Chemical), the speciﬁc inhibitor of the inducible nitric oxide synthase (iNOS),

was used to conﬁrm that the enzyme was speciﬁcally induced during the de-

scribed activation process. For these experiments, cells were stimulated with

vesicles during 16 h in the presence of 3 ␮M L-NIL and cultured with 1%

(vol/vol) Nutridoma-SP (Boehringer Mannheim) instead of FCS. To exclude the

possibility that cellular responses were affected by the presence of contaminant

LPS, control experiments were performed in the presence of puriﬁed polymyxin

B, an antibiotic that binds LPS and neutralizes its effects. Fungal vesicles and

LPS were incubated with macrophage-like cells in the presence of polymyxin B,

and nitric oxide production was evaluated. All experiments were performed in

triplicate sets and statistically analyzed by using one-way analysis of variance

(ANOVA) followed by a Bonferroni posttest.

Determination of internalization of C. neoformans by ﬂow cytometry and

ﬂuorescence microscopy. To determine internalization levels of C. neoformans

during interaction with RAW 264.7 cells, we adapted the method of Chaka et al.

(10). Brieﬂy, yeast cells were incubated with ﬂuorescein isothicyanate (FITC) at

0.5 mg/ml in PBS (25°C) for 10 min. After extensive washing with PBS, C.

1602 OLIVEIRA ET AL. INFECT.IMMUN.

by on March 18, 2010 iai.asm.orgDownloaded from

neoformans yeast cells were incubated with macrophage-like cells for3hata5:1

fungus/host cell ratio, followed by extensive washing with PBS for removal of

nonadherent fungi. The ﬂuorescence intensity of the macrophage-like cells was

therefore a function of association with FITC-labeled C. neoformans. The in-

fected cells were then detached from tissue culture plates by pipetting or scrap-

ing, ﬁxed with 4% paraformaldehyde, and analyzed in a FACSCalibur ﬂow

cytometer (BD Biosciences). The data were analyzed using winMDI 2.9 software.

The data were statistically analyzed using the one-way ANOVA test followed by

the Bonferroni posttest. To visualize the overall aspect of the cells, RAW cells

infected with FITC-labeled yeast cells were washed with PBS, sealed under a

coverslip, and visualized by epiﬂuorescence. Samples were observed with an

Axioplan 2 (Zeiss, Germany) ﬂuorescence microscope. Images were acquired

using a Color View SX digital camera and processed with the analySIS software

system (Soft Image System).

Antifungal activity of RAW 264.7 stimulated with vesicles. Macrophage mono-

layers were stimulated for 16 h with vesicles at a ﬁnal concentration correspond-

ing to 0.4 ␮g/ml sterol. The supernatant was removed and the monolayer washed

with serum-free DMEM. Aliquots of fungal cells suspended in DMEM were

added to the macrophage monolayers at a 5:1 yeast/macrophage ratio. After

incubation for3hat37°C (7.5% CO

atmosphere), the samples were washed

three times with PBS to remove nonadherent fungi; fresh medium was added,

and incubation was continued for an additional 2 or 5 h (for a total of 5 or 8 h

of infection) under the conditions described above. After incubation, cells were

again washed and then lysed with sterile cold water. Macrophage lysates con-

taining fungal cells were immediately plated onto Sabouraud agar plates for CFU

determination. Controls consisted of supernatants of nonstimulated macro-

phages cultivated under the same conditions. All experiments were performed in

triplicate sets and statistically analyzed by one-way ANOVA followed by the

Bonferroni posttest.

RESULTS

Vesicles secreted by C. neoformans are internalized by mac-

rophages. Fungal vesicles are believed to be released to the

extracellular space during in vitro macrophage infection and

animal cryptococcosis (65). However, it is unknown whether

vesicular components are incorporated by host cells and

whether these cells respond to vesicles. In this context, we ﬁrst

evaluated whether incubation of macrophages with vesicle

fractions isolated from cryptococcal supernatants would result

in their delivery into host cells. To follow internalization of

extracellular vesicles, we stained isolated vesicles with the red-

ﬂuorescent lipophilic compound dialkylcarbocyanine iodide

(DiI). Coincubation of RAW 264.7 cells performed using Dil-

labeled vesicle preparations and confocal microscopy analysis

of RAW 264.7 revealed that they were internalized by the

macrophage-like cells (Fig. 1A). Vesicles that were smaller

than 200 nm were not visualized, a result which was possibly

related to the resolution limit of ﬂuorescence microscopy. The

absence of colocalization with CtxB indicates that vesicles do

not fuse with plasma membranes. Three-dimensional recon-

struction of these cells is shown in movie S1 in the supplemen-

tal material.

We demonstrated previously that vesicles secreted by C.

neoformans also transport antigenic proteins (64). We there-

fore used serum samples from cryptococcosis patients to track

vesicle-associated antigenic components. In fact, components

showing serologic reactivity colocalized with vesicle compart-

ments labeled with DiI within macrophages (Fig. 1B). Serum

samples from healthy individuals did not react with vesicle-

treated RAW cells (data not shown).

Macrophage exposure to the vesicles for 16 h did not lead to

cell lysis (Fig. 1C), showing that, under the experimental con-

ditions used in this study, vesicular compounds do not cause

acute toxicity to these cells.

Secreted vesicles modulate NO and cytokine production by

macrophages. NO has a well-established role in the protective

responses that occur during C. neoformans infection (66). In

this context, we evaluated whether vesicles produced by encap-

sulated and acapsular C. neoformans strains would modulate

the production of NO by RAW macrophages. Vesicle fractions

from both wild-type parental and mutant strains induced

dose-dependent production of NO (Fig. 2A). However, larger

amounts of NO were produced upon stimulation with vesicles

from the acapsular mutant. In support of these data, NO pro-

duction was diminished when we treated macrophages with

vesicles produced by the acapsular mutant in the presence of

serotype A and D GXMs in comparison to macrophages sub-

jected to treatment with native vesicles from acapsular cells

(Fig. 2B). Together with the fact that vesicles from the two

encapsulated strains induced similar levels of NO (Fig. 2B),

these data suggest that different serotypes of GXM reduce

vesicle-induced NO production. The polysaccharide concentra-

tion used in this experiment was determined based on the GXM

concentration usually found in vesicles (30 ␮g/ml; data not

shown). LPS was used as a positive control (data not shown).

Under our experimental conditions, NO production was de-

pendent on the activity of induced nitric oxide synthase

(iNOS), since NO production was drastically reduced by the

presence of L-NIL (Fig. 2B). High levels of NO were detected

after exposure to vesicles in the presence of polymyxin B. In

FIG. 1. Incorporation of vesicular components by macrophages.

DiI-stained vesicles (red ﬂuorescence) were incubated with RAW

264.7 macrophages, followed by staining of the nucleus with DAPI

(blue ﬂuorescence). (A) A plasma membrane was stained with FITC-

labeled cholera toxin subunit B (green ﬂuorescence). (B) Macrophages

were incubated with serum samples from patients with cryptococcosis

followed by Alexa Fluor 488 anti-human IgG antibody. The merged

image at the right (third panel) demonstrates that DiI-stained com-

partments colocalize, at least partially, with antigenic vesicular com-

ponents (arrowheads). Scale bars, 10 ␮m. (C) Macrophage viability is

apparently not affected by the vesicles, as demonstrated by the mea-

surement of LDH activity in supernatants of macrophages treated with

vesicles (at a 2 ␮g/ml sterol concentration) or incubated in regular

medium. As a positive control for enzyme activity, the macrophages

were incubated with Triton X-100. (A

Ϫ A

)

340

nm indicates the

difference between the initial and ﬁnal absorbance readings.

OL. 78, 2010 C. NEOFORMANS EXTRACELLULAR VESICLES 1603

by on March 18, 2010 iai.asm.orgDownloaded from

contrast, the effect of LPS was completely inhibited by the

presence of polymyxin B under these conditions (data not

shown).

The response of macrophage-like cells to stimulation with

vesicles was also determined by evaluation of their ability to

produce cytokines. The cytokines analyzed were TNF-␣, IL-10,

and TGF-␤. TNF-␣ was analyzed because of its capacity to

induce the fungicidal activity of mouse peritoneal macrophages

against C. neoformans (31), while IL-10 and TGF-␤ are posi-

tively modulated by the vesicular component GXM (42, 74).

To evaluate the inﬂuence of known modulatory effects of

GXM on macrophages (74), we compared the macrophage

responses to vesicles isolated from an encapsulated C. neofor-

mans isolate (HEC3393) with the responses to those isolated

from an acapsular mutant (CAP67). Vesicle fractions from

both strains induced a signiﬁcant increase in TNF-␣ produc-

tion compared to those from nonstimulated cells (Fig. 3). It is

noteworthy that CAP67 vesicles induced an even higher pro-

duction of TNF-␣ in comparison to HEC3393 vesicles (Fig. 3),

possibly because these fractions lack GXM, which causes po-

tent depressive effects on immune function (44, 45, 70, 73).

IL-10 and TGF-␤ levels were increased following exposure to

either vesicle fraction (Fig. 3). Although levels of TGF-␤ pro-

duction by macrophages were similar in the two systems,

HEC3393 vesicles induced signiﬁcantly higher production of

IL-10 than CAP67 vesicles. These data indicate that macro-

phages differentially respond to stimulation by vesicles of C.

neoformans depending on vesicle composition.

Phagocytic and microbicidal activity of macrophages is

stimulated by secreted vesicles. We evaluated the ability of

vesicle-primed macrophages to phagocytose nonopsonized en-

capsulated C. neoformans cells. Control and vesicle-stimulated

macrophages were challenged with FITC-labeled C. neofor-

mans. Association indices were then determined by ﬂow cy-

tometry. We observed that 62% of macrophages activated with

CAP67 vesicles displayed ﬂuorescence (Fig. 4A; M1 marker)

FIG. 2. Vesicle-stimulated macrophages produce nitric oxide. (A) Dose-dependent production of NO was observed for vesicle fractions from

both acapsular and encapsulated fungi. The asterisks indicate P Ͻ 0.05. Vesicle concentrations are expressed as a function of the sterol content

in their membranes. (B) Macrophages were incubated overnight with vesicles from different strains at a ﬁnal concentration of 0.4 ␮g/ml of sterol.

GXM and L-NIL were added in ﬁnal concentrations of 30 ␮g/ml and 3 ␮M, respectively. The levels of nitric oxide production in the culture

medium (no vesicles) were below the detection limit of the technique. Bars represent averages of three measurements; brackets denote standard

deviations. Data shown are representative of the results of three independent experiments.

FIG. 3. Proﬁle of cytokine production by macrophages in response to stimulation with vesicle fractions. Macrophages were stimulated with

vesicles isolated from culture supernatants from encapsulated (HEC3393) and acapsular (CAP67) C. neoformans cells. The sterol concentration

corresponded to 0.4 ␮g/ml in all fractions. The basal production of each cytokine in the medium alone is also shown (ﬁrst bars). TNF-␣, TGF-␤,

and IL-10 concentrations in the supernatant were determined by capture ELISA. Statistical signiﬁcance values are highlighted; P Ͻ 0.05. Bars

represent averages of three measurements; brackets denote standard deviations. Data shown are representative of the results of three independent

experiments.

1604 OLIVEIRA ET AL. I

NFECT.IMMUN.

by on March 18, 2010 iai.asm.orgDownloaded from

as a consequence of infection with FITC-labeled C. neofor-

mans. Interestingly, 41% of the macrophages were positive for

FITC staining after exposure to HEC3393 vesicles. In contrast,

only 35% of control cells contained yeast cells. Although the

percentage of ﬂuorescence in M1 tended to be higher in mac-

rophages treated with HEC3393 vesicles than in control mac-

rophages, there was no statistical signiﬁcance to the difference.

Epiﬂuorescence analysis suggested that yeast internalization

was maximized under the conditions used in our experiments.

The ﬁndings were supported by differential interference con-

trast (DIC) and ﬂuorescence microscopy results (Fig. 4B).

Conﬁrming the results of ﬂuorescence-activated cell sorter

(FACS) analysis, macrophages stimulated with CAP67 vesicles

had more internalized fungi per macrophage than under any

other set of conditions (data not shown).

Since phagocytosis and production of antimicrobial com-

pounds increased after treatment with fungal vesicles, we eval-

uated the microbicidal activity of macrophages after exposing

them to vesicles from encapsulated and acapsular C. neofor-

mans strains. Control or vesicle-treated RAW cells were incu-

bated with nonopsonized C. neoformans. After 3 h, culture

ﬂuids containing unattached fungi were removed and replaced

FIG. 4. Macrophages stimulated with vesicles exhibit enhanced phagocytosis. (A) Macrophages were treated overnight with vesicles derived

from CAP67 or HEC3393 at a sterol concentration of 1 ␮M. Gray peaks represent uninfected macrophages; open histograms show the population

analysis after interaction with FITC-C. neoformans within the M1 region, and the corresponding macrophage ﬂuorescence increase is indicated

numerically. These data are representative of the results of three individual experiments. The percentages of positive ﬂuorescence (M1) are

indicated. Asterisks denote P Ͼ 0.05. (B) Analysis by ﬂuorescence microscopy of the material prepared for ﬂow cytometry. Infected cells were

visualized using differential interference contrast microscopy and ﬂuorescence microscopy. Scale bars, 10 ␮M.

OL. 78, 2010 C. NEOFORMANS EXTRACELLULAR VESICLES 1605

by on March 18, 2010 iai.asm.orgDownloaded from

by fresh media. This step was followed by an additional incu-

bation of 2 or 5 h. After these periods, supernatants were

removed and cell lysates of control and stimulated macro-

phages were assayed for the presence of viable cryptococci.

Macrophages treated with CAP67 vesicles manifested greater

killing activity than HEC3393 vesicle-activated macrophages

(Fig. 5). In fact, stimulation of macrophages with vesicles from

encapsulated yeast cells resulted in signiﬁcant microbicidal ac-

tivity only after8hofincubation. Although we cannot ensure

that the only difference between the HEC3393 and CAP67

vesicles was the presence of GXM, it is conceivable that the

less efﬁcient fungicidal activity observed of macrophages

treated with HEC3393 was related to the fact that GXM neg-

atively modulates the macrophage response against C. neofor-

mans (45, 73). Albeit a tendency of growth inhibition was

visualized on control macrophages after8hofincubation, it is

important that no statistical signiﬁcance difference was ob-

served compared to the results obtained after5hofincuba-

tion.

DISCUSSION

The host response to microbial antigens is a complex process

that is inﬂuenced by a number of factors, including the con-

centration of immunogens, the efﬁcacy of exposure of these

molecules to effector cells, and the ability of the infectious

agent to produce and secrete immunologically active com-

pounds. Consequently, the outcome of the interaction of in-

fectious agents with host cells includes multiple possibilities,

such as the persistence of the microbe within host tissues,

causing cell damage, and the limitation of microbial coloniza-

tion, resulting in disease control (8).

It was suggested that fungal cells secrete vesicles during

interaction with phagocytes in vivo (65). Therefore, one might

expect that the concentration of such a complex array of mol-

ecules in vesicular structures could directly inﬂuence the inter-

action of fungal pathogens with their hosts. Supporting this

hypothesis, Gram-negative bacteria have been shown to con-

stitutively secrete outer membrane vesicles composed of lipo-

polysaccharide, phospholipids, outer membrane proteins, and

soluble periplasmic proteins, most of which are important vir-

ulence determinants (5, 36). This secretion mechanism is

thought to be employed by bacteria to deliver virulence factors

in the host cell (29). Recently, Lee and colleagues demon-

strated that Gram-positive bacteria also secrete vesicles, sug-

gesting that this process could be involved with protein transfer

to other bacteria and is conserved among different organisms

(38). Although the origin of fungal extracellular vesicles stud-

ied by our group is very different from that of their bacterial

counterparts, we investigated whether cryptococcal vesicles

also affect host-pathogen interactions.

In the present work we demonstrate that extracellular se-

creted vesicles from pathogenic fungi directly modulate host-

pathogen interactions and propose a model for these interac-

tions. According to this model, differences in vesicle numbers

and composition, as well as in the presence of GXM, could

affect the outcome of the C. neoformans-macrophage interac-

tion by affecting the state of macrophage activation. Strongly

suggestive evidence that vesicles are made in tissue comes from

electron microscopy and the observation of a serum antibody

response to vesicle components in infected mice (64, 65). Al-

though our work indicates that comparable effects could occur

in vivo, such studies remain to be done, and currently there are

formidable technical hurdles interfering with the demonstra-

tion of in vivo effects. Nevertheless, our results are likely to

have anticipated the observation of extracellular vesicles as

important modulators of infection outcome and participation

in the activation of immune response, as recently described for

mammalian exosomes (69).

C. neoformans produces several exocellular antigens with

apparently conﬂicting functions with regard to pathogenesis.

For instance, GXM, the major cryptococcal virulence factor,

protects the fungus against phagocytosis and inhibits leukocyte

migration (17, 21, 22, 34). However, this polysaccharide, in its

capsular form, is also an efﬁcient inducer of the activation of

the alternative pathway of the complement system, which re-

sults in the deposition of opsonins in the capsule and increased

phagocytosis (33, 35). Another example of apparently oppos-

ing functions of capsular components of C. neoformans in-

volves the soluble forms of GXM and GalXM. Both of these

well-known capsular components activate inducible nitric ox-

ide synthase and the consequent production of nitric oxide in

macrophages (74). However, a heavily encapsulated cryptococ-

cal strain suppressed the production of this antimicrobial com-

pound in CpG-oligodeoxynucleotide-stimulated macrophages

(78), which conﬁrms previous studies showing that C. neofor-

mans cells fail to induce nitric oxide synthase in primed murine

macrophage-like cells (52). These results reveal the enormous

complexity of the events involved in the interaction of C. neo-

formans with phagocytes and the difﬁculty in predicting which

pathogenesis-related structure will have the greatest effect dur-

ing infection of host cells. The cryptococcal vesicles contain at

least 81 different fungal molecules with potentially different

functional impacts on immunity (64, 65). Therefore, the re-

sponse of host cells to vesicle preparations is virtually unpre-

dictable, which led us to experimentally address some of the

functions of macrophages after their exposure to vesicle frac-

tions of C. neoformans.

FIG. 5. Fungicidal activity of vesicle-stimulated macrophages.

Macrophages treated with vesicles isolated from encapsulated or acap-

sular C. neoformans were incubated with the encapsulated C. neofor-

mans strain for different periods for CFU determination of numbers of

intracellular C. neoformans. CFU values were multiplied using the

dilution factor to generate absolute cell numbers. Bars represent

averages of three measurements; brackets represent standard devi-

ations. Statistically signiﬁcant differences are highlighted.

1606 OLIVEIRA ET AL. I

NFECT.IMMUN.

by on March 18, 2010 iai.asm.orgDownloaded from

As recently reported for bacterial vesicles (5), our results

indicate that macrophages are able to internalize vesicular

components. Exposure of macrophages to vesicle preparations

resulted in enhanced levels of IL-10 and TGF-␤, anti-inﬂam-

matory cytokines whose production is positively modulated by

the vesicular component GXM (11, 42). In agreement with the

role of GXM in the upregulation of IL-10, CAP67 vesicles

induced a signiﬁcantly lower amount of this cytokine, although

the level was signiﬁcantly greater than in untreated macro-

phages. The increased production of IL-10 was recently asso-

ciated with a protective immune response to cryptococcosis,

which was stimulated by a peptide mimotope of GXM (13).

This higher efﬁciency was inferred from (i) the lower concen-

tration of CAP67 vesicles required for induction of high levels

of NO, (ii) the enhanced ability to induce yeast internalization,

and (iii) the requirement of a shorter incubation period to

display potent microbicidal activity.

Transmission electron microscopy demonstrated that C.

neoformans vesicle fractions contain subpopulations that differ

in morphological characteristics, suggesting the existence of

different biosynthetic steps for vesicle biogenesis (62). More

importantly, the detection of different morphological types

suggests that vesicles of diverse molecular compositions occur.

In a recent report, we used gradient fractionation of vesicles to

demonstrate that GXM concentrations differ considerably in

fractions with different densities (57). The methods currently

used for vesicle puriﬁcation do not discriminate between ves-

icles of different characteristics, resulting in heterogeneous

preparations. Here we showed that differences in molecular

composition of vesicles between two strains were translated

into functional differences in the modulation of host cell

functions. Although this vesicle heterogeneity complicates the

functional analysis of speciﬁc vesicle populations, it approxi-

mates the reality that C. neoformans simultaneously secretes

different vesicle types (57). Assuming that C. neoformans se-

cretes vesicles during interaction with host cells (65), the over-

all effects of the exposure of phagocytes to extracellular frac-

tions could be better reﬂected in experiments using “crude”

vesicle samples, although macrophage function could be dif-

ferentially affected by distinct vesicle fractions.

Although our results provide strong evidence that crypto-

coccal vesicle preparations stimulate macrophage-like cells to

alter their cytokine production and enhance their antifungal

function, there are some experimental limitations that pre-

clude more generalized conclusions at this time. Currently we

do not have a reliable method to accurately enumerate vesicles

in our preparations and consequently cannot estimate the

number of vesicles required for the activation effect. It is con-

ceivable, and probably likely, that the effect of the presence of

vesicles on macrophages depends on the number of vesicles

that interact with each phagocytic cell such that, at low vesicle/

macrophage ratios, the effect results in activation whereas, at

higher ratios, the effect is detrimental to the macrophage. The

in vivo milieu may also impact the interactions; for instance,

secreted host lipases could disrupt the vesicles, leading to ear-

lier release of vesicular contents. Furthermore, the impact of

vesicles produced by intracellular fungi may be different from

that of extracellular vesicle preparations. Notwithstanding

these uncertainties, the results further support our conclusion

that fungal vesicles are biologically active; this information

opens new venues of investigation in fungal-host cell interac-

tions.

In this report, we demonstrate that the recently described

extracellular fungal vesicles can modify the functions of mam-

malian cells, altering the fate of C. neoformans after phagocy-

tosis by macrophages. These results strongly suggest the po-

tential of these vesicular structures to modify the course of

cryptococcal infections through their effects on host phagocytic

cells. Fungal vesicles are apparently related to mammalian

exosomes (58, 64). In this regard, it is noteworthy that exosome

preparations from dendritic cells are being investigated as po-

tential anticancer vaccines (26). The current results and the

fact that vesicle-related proteins are immunogenic (1, 62, 64)

suggest that vesicle preparations and/or their components

could have utility as fungal vaccines, which will be a focus of

future studies in our laboratory.

ACKNOWLEDGMENTS

L.N. and M.L.R. are supported by grants from the Brazilian agencies

CNPq, FAPERJ, and FAPESP. L.N. was supported in part by an

Interhemispheric Research Training Grant in Infectious Diseases,

Fogarty International Center (NIH D43-TW007129). J.D.N. was sup-

ported in part by NIH RO1 AI052733. A.C. was supported by NIH

awards AI033774, HL059842, and 2R37AI033142. C.G.F.-D.-L. is sup-

ported by grants from CNPq, FAPERJ, and the National Institute of

Science and Technology in Vaccines (INCTV).

D.L.O. is a Ph.D. student at Instituto de Bioquímica Me´dica,

UFRJ.

We thank the AECOM Analytical Imaging Facility for help with

confocal experiments. We also thank Jorge Jose´Jo´ B. Ferreira for

helpful discussions.

REFERENCES

1. Albuquerque, P. C., E. S. Nakayasu, M. L. Rodrigues, S. Frases, A. Casade-

vall, R. M. Zancope-Oliveira, I. C. Almeida, and J. D. Nosanchuk. 2008.

Vesicular transport in Histoplasma capsulatum: an effective mechanism for

trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol.

10:1695–1710.

2. Alvarez, M., and A. Casadevall. 2007. Cell-to-cell spread and massive vacu-

ole formation after Cryptococcus neoformans infection of murine macro-

phages. BMC Immunol. 8:16.

3. Alvarez, M., and A. Casadevall. 2006. Phagosome extrusion and host-cell

survival after Cryptococcus neoformans phagocytosis by macrophages. Curr.

Biol. 16:2161–2165.

4. Beauvais, A., M. Monod, J. Wyniger, J. P. Debeaupuis, E. Grouzmann, N.

Brakch, J. Svab, A. G. Hovanessian, and J. P. Latge. 1997. Dipeptidyl-

peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to hu-

mans. Infect. Immun. 65:3042–3047.

5. Bomberger, J. M., D. P. Maceachran, B. A. Coutermarsh, S. Ye, G. A.

O’Toole, and B. A. Stanton. 2009. Long-distance delivery of bacterial viru-

lence factors by Pseudomonas aeruginosa outer membrane vesicles. PLoS

Pathog. 5:e1000382.

6. Bulmer, G. S., and M. D. Sans. 1968. Cryptococcus neoformans. III. Inhibi-

tion of phagocytosis. J. Bacteriol. 95:5–8.

7. Casadevall, A., J. D. Nosanchuk, P. Williamson, and M. L. Rodrigues. 2009.

Vesicular transport across the fungal cell wall. Trends Microbiol. 17:158–

162.

8. Casadevall, A., and L. Pirofski. 2001. Host-pathogen interactions: the at-

tributes of virulence. J. Infect. Dis. 184:337–344.

9. Chafﬁn, W. L., J. L. Lopez-Ribot, M. Casanova, D. Gozalbo, and J. P.

Martinez. 1998. Cell wall and secreted proteins of Candida albicans: identi-

ﬁcation, function, and expression. Microbiol. Mol. Biol. Rev. 62:130–180.

10. Chaka, W., J. Scharringa, A. F. Verheul, J. Verhoef, A. G. Van Strijp, and

I. M. Hoepelman. 1995. Quantitative analysis of phagocytosis and killing of

Cryptococcus neoformans by human peripheral blood mononuclear cells by

ﬂow cytometry. Clin. Diagn. Lab Immunol. 2:753–759.

11. Chiapello, L. S., J. L. Baronetti, M. P. Aoki, S. Gea, H. Rubinstein, and D. T.

Masih. 2004. Immunosuppression, interleukin-10 synthesis and apoptosis are

induced in rats inoculated with Cryptococcus neoformans glucuronoxyloman-

nan. Immunology 113:392–400.

12. Cox, G. M., J. Mukherjee, G. T. Cole, A. Casadevall, and J. R. Perfect. 2000.

Urease as a virulence factor in experimental cryptococcosis. Infect. Immun.

68:443–448.

VOL. 78, 2010 C. NEOFORMANS EXTRACELLULAR VESICLES 1607

by on March 18, 2010 iai.asm.orgDownloaded from

13. Datta, K., A. Lees, and L. A. Pirofski. 2008. Therapeutic efﬁcacy of a con-

jugate vaccine containing a peptide mimotope of cryptococcal capsular poly-

saccharide glucuronoxylomannan. Clin. Vaccine Immunol. 15:1176–1187.

14. De Jesus, M., A. M. Nicola, M. L. Rodrigues, G. Janbon, and A. Casadevall.

2009. Capsular localization of the Cryptococcus neoformans polysaccharide

component galactoxylomannan. Eukaryot. Cell 8:96–103.

15. Del Poeta, M. 2004. Role of phagocytosis in the virulence of Cryptococcus

neoformans. Eukaryot. Cell 3:1067–1075.

16. Djordjevic, J. T., M. Del Poeta, T. C. Sorrell, K. M. Turner, and L. C.

Wright. 2005. Secretion of cryptococcal phospholipase B1 (PLB1) is regu-

lated by a glycosylphosphatidylinositol (GPI) anchor. Biochem. J. 389:803–

812.

17. Dong, Z. M., and J. W. Murphy. 1997. Cryptococcal polysaccharides bind to

CD18 on human neutrophils. Infect. Immun. 65:557–563.

18. Douwes, J., P. Versloot, A. Hollander, D. Heederik, and G. Doekes. 1995.

Inﬂuence of various dust sampling and extraction methods on the measure-

ment of airborne endotoxin. Appl. Environ. Microbiol. 61:1763–1769.

19. Eigenheer, R. A., Y. Jin Lee, E. Blumwald, B. S. Phinney, and A. Gelli. 2007.

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and pro-

teases of Cryptococcus neoformans. FEMS Yeast Res. 7:499–510.

20. Eisenman, H. C., S. Frases, A. M. Nicola, M. L. Rodrigues, and A. Casade-

vall. 2009. Vesicle-associated melanization in Cryptococcus neoformans. Mi-

crobiology 155:3860–3867.

21. Ellerbroek, P. M., D. J. Lefeber, R. van Veghel, J. Scharringa, E. Brouwer,

G. J. Gerwig, G. Janbon, A. I. Hoepelman, and F. E. Coenjaerts. 2004.

O-Acetylation of cryptococcal capsular glucuronoxylomannan is essential for

interference with neutrophil migration. J. Immunol. 173:7513–7520.

22. Ellerbroek, P. M., L. H. Ulfman, A. I. Hoepelman, and F. E. Coenjaerts.

2004. Cryptococcal glucuronoxylomannan interferes with neutrophil rolling

on the endothelium. Cell Microbiol. 6:581–592.

23. Feldmesser, M., S. Tucker, and A. Casadevall. 2001. Intracellular parasitism

of macrophages by Cryptococcus neoformans. Trends Microbiol. 9:273–278.

24. Ghosn, E. E., M. Russo, and S. R. Almeida. 2006. Nitric oxide-dependent

killing of Cryptococcus neoformans by B-1-derived mononuclear phagocyte.

J. Leukoc. Biol. 80:36–44.

25. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and

S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N]nitrate in

biological ﬂuids. Anal. Biochem. 126:131–138.

26. Hao, S., T. Moyana, and J. Xiang. 2007. Cancer immunotherapy by exosome-

based vaccines. Cancer Biother. Radiopharm. 22:692–703.

27. Jacobson, E. S. 2000. Pathogenic roles for fungal melanins. Clin. Microbiol.

Rev. 13:708–717.

28. Jacobson, E. S., D. J. Ayers, A. C. Harrell, and C. C. Nicholas. 1982. Genetic

and phenotypic characterization of capsule mutants of Cryptococcus neofor-

mans. J. Bacteriol. 150:1292–1296.

29. Kadurugamuwa, J. L., and T. J. Beveridge. 1995. Virulence factors are

released from Pseudomonas aeruginosa in association with membrane vesi-

cles during normal growth and exposure to gentamicin: a novel mechanism

of enzyme secretion. J. Bacteriol. 177:3998–4008.

30. Kawakami, K., Y. Koguchi, M. H. Qureshi, S. Yara, Y. Kinjo, K. Uezu, and

A. Saito. 2000. NK cells eliminate Cryptococcus neoformans by potentiating

the fungicidal activity of macrophages rather than by directly killing them

upon stimulation with IL-12 and IL-18. Microbiol. Immunol. 44:1043–1050.

31. Kawakami, K., M. H. Qureshi, Y. Koguchi, T. Zhang, H. Okamura, M.

Kurimoto, and A. Saito. 1999. Role of TNF-alpha in the induction of fun-

gicidal activity of mouse peritoneal exudate cells against Cryptococcus neo-

formans by IL-12 and IL-18. Cell Immunol. 193:9–16.

32. Kozel, T. R., B. Highison, and C. J. Stratton. 1984. Localization on encap-

sulated Cryptococcus neoformans of serum components opsonic for phago-

cytosis by macrophages and neutrophils. Infect. Immun. 43:574–579.

33. Kozel, T. R., and G. S. Pfrommer. 1986. Activation of the complement

system by Cryptococcus neoformans leads to binding of iC3b to the yeast.

Infect. Immun. 52:1–5.

34. Kozel, T. R., G. S. Pfrommer, A. S. Guerlain, B. A. Highison, and G. J.

Highison. 1988. Role of the capsule in phagocytosis of Cryptococcus neofor-

mans. Rev. Infect. Dis. 10(Suppl. 2):S436–S439.

35. Kozel, T. R., M. A. Wilson, G. S. Pfrommer, and A. M. Schlageter. 1989.

Activation and binding of opsonic fragments of C3 on encapsulated Crypto-

coccus neoformans by using an alternative complement pathway reconsti-

tuted from six isolated proteins. Infect. Immun. 57:1922–1927.

36. Kuehn, M. J., and N. C. Kesty. 2005. Bacterial outer membrane vesicles and

the host-pathogen interaction. Genes Dev. 19:2645–2655.

37. Latge´, J. P., H. Kobayashi, J. P. Debeaupuis, M. Diaquin, J. Sarfati, J. M.

Wieruszeski, E. Parra, J. P. Bouchara, and B. Fournet. 1994. Chemical and

immunological characterization of the extracellular galactomannan of As-

pergillus fumigatus. Infect. Immun. 62:5424–5433.

38. Lee, E. Y., D. Y. Choi, D. K. Kim, J. W. Kim, J. O. Park, S. Kim, S. H. Kim,

D. M. Desiderio, Y. K. Kim, K. P. Kim, and Y. S. Gho. 2009. Gram-positive

bacteria produce membrane vesicles: proteomics-based characterization of

Staphylococcus aureus-derived membrane vesicles. Proteomics 9:5425–5436.

39. Liu, L., R. P. Tewari, and P. R. Williamson. 1999. Laccase protects Crypto-

coccus neoformans from antifungal activity of alveolar macrophages. Infect.

Immun. 67:6034–6039.

40. Ma, H., J. E. Croudace, D. A. Lammas, and R. C. May. 2007. Direct cell-

to-cell spread of a pathogenic yeast. BMC Immunol. 8:15.

41. Ma, H., J. E. Croudace, D. A. Lammas, and R. C. May. 2006. Expulsion of

live pathogenic yeast by macrophages. Curr. Biol. 16:2156–2160.

42. Mariano Andrade, R., G. Monteiro Almeida, G. Alexandre DosReis, and C.

Alves Melo Bento. 2003. Glucuronoxylomannan of Cryptococcus neoformans

exacerbates in vitro yeast cell growth by interleukin 10-dependent inhibition

of CD4

T lymphocyte responses. Cell Immunol. 222:116–125.

43. Miller, M. F., and T. G. Mitchell. 1991. Killing of Cryptococcus neoformans

strains by human neutrophils and monocytes. Infect. Immun. 59:24–28.

44. Mirshaﬁey, A., D. T. Ngoc Dzung, J. W. Murphy, M. R. Khorramizadeh, F.

Saadat, F. Mehrabian, and P. Larsson. 2004. Tolerability and anti-inﬂam-

matory effects of glucuronoxylomannan in collagen-induced arthritis. Scand.

J. Immunol. 60:226–232.

45. Monari, C., F. Bistoni, A. Casadevall, E. Pericolini, D. Pietrella, T. R. Kozel,

and A. Vecchiarelli. 2005. Glucuronoxylomannan, a microbial compound,

regulates expression of costimulatory molecules and production of cytokines

in macrophages. J. Infect. Dis. 191:127–137.

46. Monari, C., A. Casadevall, C. Retini, F. Baldelli, F. Bistoni, and A. Vec-

chiarelli. 1999. Antibody to capsular polysaccharide enhances the function of

neutrophils from patients with AIDS against Cryptococcus neoformans.

AIDS 13:653–660.

47. Monari, C., T. R. Kozel, F. Bistoni, and A. Vecchiarelli. 2002. Modulation of

C5aR expression on human neutrophils by encapsulated and acapsular Cryp-

tococcus neoformans. Infect. Immun. 70:3363–3370.

48. Monari, C., E. Pericolini, G. Bistoni, A. Casadevall, T. R. Kozel, and A.

Vecchiarelli. 2005. Cryptococcus neoformans capsular glucuronoxylomannan

induces expression of Fas ligand in macrophages. J. Immunol. 174:3461–

3468.

49. Monod, M., K. Jaton-Ogay, and U. Reichard. 1999. Aspergillus fumigatus-

secreted proteases as antigenic molecules and virulence factors. Contrib.

Microbiol. 2:182–192.

50. Mukherjee, S., M. Feldmesser, and A. Casadevall. 1996. J774 murine mac-

rophage-like cell interactions with Cryptococcus neoformans in the presence

and absence of opsonins. J. Infect. Dis. 173:1222–1231.

51. Murphy, J. W., F. Schafer, A. Casadevall, and A. Adesina. 1998. Antigen-

induced protective and nonprotective cell-mediated immune components

against Cryptococcus neoformans. Infect. Immun. 66:2632–2639.

52. Naslund, P. K., W. C. Miller, and D. L. Granger. 1995. Cryptococcus neo-

formans fails to induce nitric oxide synthase in primed murine macrophage-

like cells. Infect. Immun. 63:1298–1304.

53. Nichols, K. L., S. K. Bauman, F. B. Schafer, and J. W. Murphy. 2002.

Differences in components at delayed-type hypersensitivity reaction sites in

mice immunized with either a protective or a nonprotective immunogen of

Cryptococcus neoformans. Infect. Immun. 70:591–600.

54. Nicola, A. M., A. Casadevall, and D. L. Goldman. 2008. Fungal killing by

mammalian phagocytic cells. Curr. Opin. Microbiol. 11:313–317.

55. Nicola, A. M., S. Frases, and A. Casadevall. 2009. Lipophilic dye staining of

Cryptococcus neoformans extracellular vesicles and capsule. Eukaryot. Cell

8:1373–1380.

56. Nosanchuk, J. D., L. Nimrichter, A. Casadevall, and M. L. Rodrigues. 2008.

A role for vesicular transport of macromolecules across cell walls in fungal

pathogenesis. Commun. Integr. Biol. 1:37–39.

57. Oliveira, D. L., L. Nimrichter, K. Miranda, S. Frases, K. F. Faull, A. Cas-

adevall, and M. L. Rodrigues. 2009. Cryptococcus neoformans cryoimmuno-

electronmicroscopy and vesicle fractionation reveals an intimate association

between membrane lipids and glucuronxylomannan. Fungal Genet. Biol.

46:956–963.

58. Panepinto, J., K. Komperda, S. Frases, Y. D. Park, J. T. Djordjevic, A.

Casadevall, and P. R. Williamson. 2009. Sec6-dependent sorting of fungal

extracellular exosomes and laccase of Cryptococcus neoformans. Mol. Micro-

biol. 71:1165–1176.

59. Pietrella, D., C. Monari, C. Retini, B. Palazzetti, T. R. Kozel, and A. Vec-

chiarelli. 1998. Cryptococcus neoformans and Candida albicans regulate CD4

expression on human monocytes. J. Infect. Dis. 178:1464–1471.

60. Puccia, R., S. Schenkman, P. A. Gorin, and L. R. Travassos. 1986. Exocel-

lular components of Paracoccidioides brasiliensis: identiﬁcation of a speciﬁc

antigen. Infect. Immun. 53:199–206.

61. Reiss, E., M. Huppert, and R. Cherniak. 1985. Characterization of protein

and mannan polysaccharide antigens of yeasts, moulds, and actinomycetes.

Curr. Top. Med. Mycol. 1:172–207.

62. Rodrigues, M. L., M. Alvarez, F. L. Fonseca, and A. Casadevall. 2008.

Binding of the wheat germ lectin to Cryptococcus neoformans suggests an

association of chitinlike structures with yeast budding and capsular glucu-

ronoxylomannan. Eukaryot. Cell 7:602–609.

63. Rodrigues, M. L., C. S. Alviano, and L. R. Travassos. 1999. Pathogenicity of

Cryptococcus neoformans: virulence factors and immunological mechanisms.

Microbes Infect. 1:293–301.

64. Rodrigues, M. L., E. S. Nakayasu, D. L. Oliveira, L. Nimrichter, J. D.

Nosanchuk, I. C. Almeida, and A. Casadevall. 2008. Extracellular vesicles

1608 OLIVEIRA ET AL. INFECT.IMMUN.

by on March 18, 2010 iai.asm.orgDownloaded from

produced by Cryptococcus neoformans contain protein components associ-

ated with virulence. Eukaryot. Cell 7:58–67.

65. Rodrigues, M. L., L. Nimrichter, D. L. Oliveira, S. Frases, K. Miranda, O.

Zaragoza, M. Alvarez, A. Nakouzi, M. Feldmesser, and A. Casadevall. 2007.

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic

solution to the problem of fungal trans-cell wall transport. Eukaryot. Cell

6:48–59.

66. Rossi, G. R., L. A. Cervi, M. M. Garcia, L. S. Chiapello, D. A. Sastre, and

D. T. Masih. 1999. Involvement of nitric oxide in protecting mechanism

during experimental cryptococcosis. Clin. Immunol. 90:256–265.

67. Schaller, M., M. Bein, H. C. Korting, S. Baur, G. Hamm, M. Monod, S.

Beinhauer, and B. Hube. 2003. The secreted aspartyl proteinases Sap1 and

Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on

reconstituted human vaginal epithelium. Infect. Immun. 71:3227–3234.

68. Syme, R. M., J. C. Spurrell, E. K. Amankwah, F. H. Green, and C. H. Mody.

2002. Primary dendritic cells phagocytose Cryptococcus neoformans via man-

nose receptors and Fcgamma receptor II for presentation to T lymphocytes.

Infect. Immun. 70:5972–5981.

69. The´ry, C., M. Ostrowski, and E. Segura. 2009. Membrane vesicles as con-

veyors of immune responses. Nat. Rev. Immunol. 9:581–593.

70. Tissi, L., M. Puliti, F. Bistoni, P. Mosci, T. R. Kozel, and A. Vecchiarelli.

2004. Glucuronoxylomannan, the major capsular polysaccharide of Crypto-

coccus neoformans, inhibits the progression of group B streptococcal arthri-

tis. Infect. Immun. 72:6367–6372.

71. Tucker, S. C., and A. Casadevall. 2002. Replication of Cryptococcus neofor-

mans in macrophages is accompanied by phagosomal permeabilization and

accumulation of vesicles containing polysaccharide in the cytoplasm. Proc.

Natl. Acad. Sci. USA 99:3165–3170.

72. Vecchiarelli, A., D. Pietrella, P. Lupo, F. Bistoni, D. C. McFadden, and A.

Casadevall. 2003. The polysaccharide capsule of Cryptococcus neoformans

interferes with human dendritic cell maturation and activation. J. Leukoc.

Biol. 74:370–378.

73. Vecchiarelli, A., C. Retini, D. Pietrella, C. Monari, C. Tascini, T. Beccari,

and T. R. Kozel. 1995. Downregulation by cryptococcal polysaccharide of

tumor necrosis factor alpha and interleukin-1 beta secretion from human

monocytes. Infect. Immun. 63:2919–2923.

74. Villena, S. N., R. O. Pinheiro, C. S. Pinheiro, M. P. Nunes, C. M. Takiya,

G. A. DosReis, J. O. Previato, L. Mendonca-Previato, and C. G. Freire-de-

Lima. 2008. Capsular polysaccharides galactoxylomannan and glucuronoxy-

lomannan from Cryptococcus neoformans induce macrophage apoptosis me-

diated by Fas ligand. Cell Microbiol. 10:1274–1285.

75. Vyas, K. A., H. V. Patel, A. A. Vyas, and R. L. Schnaar. 2001. Segregation of

gangliosides GM1 and GD3 on cell membranes, isolated membrane rafts,

and deﬁned supported lipid monolayers. Biol. Chem. 382:241–250.

76. Wang, Y., P. Aisen, and A. Casadevall. 1995. Cryptococcus neoformans mel-

anin and virulence: mechanism of action. Infect. Immun. 63:3131–3136.

77. Wozniak, K. L., and S. M. Levitz. 2008. Cryptococcus neoformans enters the

endolysosomal pathway of dendritic cells and is killed by lysosomal compo-

nents. Infect. Immun. 76:4764–4771.

78. Xiao, G., A. Miyazato, K. Inden, K. Nakamura, K. Shiratori, K. Nakagawa,

T. Miyazawa, K. Suzuki, M. Kaku, and K. Kawakami. 2008. Cryptococcus

neoformans inhibits nitric oxide synthesis caused by CpG-oligodeoxynucle-

otide-stimulated macrophages in a fashion independent of capsular polysac-

charides. Microbiol. Immunol. 52:171–179.

79. Yauch, L. E., J. S. Lam, and S. M. Levitz. 2006. Direct inhibition of T-cell

responses by the Cryptococcus capsular polysaccharide glucuronoxyloman-

nan. PLoS Pathog. 2:e120.

80. Zaragoza, O., C. J. Chrisman, M. V. Castelli, S. Frases, M. Cuenca-Estrella,

J. L. Rodriguez-Tudela, and A. Casadevall. 2008. Capsule enlargement in

Cryptococcus neoformans confers resistance to oxidative stress suggesting a

mechanism for intracellular survival. Cell Microbiol. 10:2043–2057.

81. Zaragoza, O., M. L. Rodrigues, M. De Jesus, S. Frases, E. Dadachova, and

A. Casadevall. 2009. The capsule of the fungal pathogen Cryptococcus neo-

formans. Adv. Appl. Microbiol. 68:133–216.

82. Zaragoza, O., C. P. Taborda, and A. Casadevall. 2003. The efﬁcacy of

complement-mediated phagocytosis of Cryptococcus neoformans is depen-

dent on the location of C3 in the polysaccharide capsule and involves both

direct and indirect C3-mediated interactions. Eur. J. Immunol. 33:1957–

1967.

Editor: G. S. Deepe, Jr.

VOL. 78, 2010 C. NEOFORMANS EXTRACELLULAR VESICLES 1609

by on March 18, 2010 iai.asm.orgDownloaded from

Anexo 4:

Characterization of Yeast Extracellular Vesicles: Evidence

for the Participation of Different Pathways of Cellular

Traffic in Vesicle Biogenesis

bora L. Oliveira

, Ernesto S. Nakayasu

2.¤

, Luna S. Joffe

, Allan J. Guimara

, Tiago J. P. Sobreira

Joshua D. Nosanchuk

3,5

, Radames J. B. Cordero

, Susana Frases

, Arturo Casadevall

3,5

,IgorC.

Almeida

, Leonardo Nimrichter

, Marcio L. Rodrigues

1 Laborato

rio de Estudos Integrados em Bioquı

mica Microbiana, Instituto de Microbiologia Professor Paulo de Go

es, Rio de Janeiro, Rio de Janeiro, Brazil, 2 Department of

Biological Sciences, The Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas, United States of America, 3 Department of Medicine, Albert

Einstein College of Medicine, Bronx, New York, United States of America, 4 Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of Sa

Paulo, Sa

o Paulo, Sa

o Paulo, Brazil, 5 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America,

6 Laborato

rio de Biotecnologia, Instituto Nacional de Metrologia, Normalizac¸a

o e Qualidade Industrial, Rio de Janeiro, Rio de Janeiro, Brazil

Abstract

Background:

Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens,

these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular

space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.

Methodology/Principal Findings:

We characterized extracellular vesicle production in wild type (WT) and mutant strains of

the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering

analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-

associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB)

formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100–300 nm range were found in extracellular

fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants

with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma

membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed

a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in

both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle

production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had

slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in

vesicle fractions in comparison with WT cells.

Conclusions/Significance:

Our results suggest that both conventional and unconventional pathways of secretion are

required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast

cells.

Citation: Oliveira DL, Nakayasu ES, Joffe LS, Guimara

es AJ, Sobreira TJP, et al. (2010) Characterization of Yeast Extracellular Vesicles: Evidence for the Participation

of Different Pathways of Cellular Traffic in Vesicle Biogenesis. PLoS ONE 5(6): e11113. doi:10.1371/journal.pone.0011113

Editor: Michael Otto, National Institutes of Health, United States of America

Received March 24, 2010; Accepted May 21, 2010; Published June 14, 2010

Copyright: ß 2010 Oliveira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: LN and MLR are supported by grants from the Brazilian agencies Conselho Nacional de Desenvolvimento Cientı

fico e Tecnolo

gico (CNPq), Fundac¸a

ode

Amparo a

Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenac¸a

o de Aperfeic¸oamento de Pessoal de Nı

vel Superior (CAPES). AC is supported by NIH

awards HL059842, AI033774, AI052733, AI033142. AJG and LN were supported in part by an Interhemispheric Research Training Grant in Infectious Diseases,

Fogarty International Center (NIH D43-TW007129). JDN is supported in part by NIH AI52733-06A1 and AI056070-01A2, and a Hirschl/Weill-Caulier Career Scientist

Award. RJBC was supported by the Training Program in Cellular and Molecular 414 Biology and Genetics, T32 GM007491. ICA was partially supported by the NIH/

NCRR grant 5G12RR008124-16A1. ESN was partially supported by the George A. Krutilek memorial graduate scholarship from Graduate School, University of Texas

at El-Paso (UTEP). TJPS was supported by Fundac¸a

o de Amparo a

Pesquisa do Estado de Sa

o Paulo (FAPESP), Brazil. The authors thank the Biomolecule Analysis

Core Facility, Border Biomedical Research Center/Biology/University of Texas at El-Paso (NIH grants #5G12RR008124-16A1 and 5G12RR008124-16A1S1), for the

access to the LC-MS instrumentation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: marcio@micro.ufrj.br

. These authors contributed equally to this work.

" These authors also contributed equally to this work.

¤ Current address: Pacific Northwest National Laboratory, Richland, Washington, United States of America

PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11113

Introduction

Protein secretion is a complex process that involves many

organelles and accessory molecules. In eukaryotic cells, the most

well-studied pathway of protein secretion involves vesicular

migration from the endoplasmic reticulum to the trans face of

the Golgi and then loading into a complex network of vesicles, the

trans-Golgi reticulum [1]. The vesicular post-Golgi network is the

most prominent example of conventional mechanism of protein

secretion. These proteins are sorted in the trans-Golgi network into

transport vesicles that immediately move to and fuse with the

plasma membrane, releasing their contents by exocytosis [1].

There are multiple unconventional mechanisms of protein

secretion in eukaryotes [2]. One of these mechanisms requires

the formation of the exosomes, which are vesicles derived from

membrane invagination within endocytic compartments (endo-

somes). The formation of internal vesicles in the lumen of

endosomes generates the multivesicular bodies (MVBs), which can

fuse with the plasma membrane, resulting in the release of internal

vesicles to the extracellular milieu as exosomes [3]. Extracellular

vesicle formation could also require other cellular pathways, as

suggested for Dictyostelium discoideum. In this organism, it has been

hypothesized that the Golgi reassembly stacking protein (GRASP),

which is attached peripherally to the cytoplasmic surface of Golgi

membranes, is required for the vesicular release of acyl-CoA

binding protein [4].

Fungal cells secrete molecules of different chemical natures and

molecular masses. As typical eukaryotic organisms, fungal cells use

conventional pathways of secretion involving post-Golgi vesicles

that fuse with the plasma membrane to release their cargo [5,6]. In

fact, it is well known that yeast cells continuously secrete a number

of enzymes that remain localized in the periplasm [7] but, until

recently, trans-cell wall secretion in fungi is a relatively unknown

cellular event, which consequently, had not been extensively

studied. Over the past few years, the existence of trans-cell wall

transport of intact vesicles has been reported and partially

characterized in pathogenic and non-pathogenic species of fungi

[8,9,10,11,12,13,14,15]. These vesicles contain a number of

proteins, lipids, polysaccharides and pigments of a wide molecular

mass range [8,11,14,15]. Therefore, we have proposed that

extracellular vesicle secretion represents a eukaryotic solution to

the problem of trans-cell wall transport, especially for large

molecules [9,12].

Although fungal extracellular vesicles have been generally

termed ‘fungal exosomes’ [9,14,16], different studies suggest that

their release to the extracellular space requires elements of the

conventional post-Golgi secretory pathway [16,17]. Yoneda and

Doering demonstrated that a Cryptococcus neoformans strain defective

in the production of Sav1p, a homolog of the S. cerevisiae small

GTPase Sec4p, accumulates post-Golgi vesicles under restrictive

conditions [17], a morphological feature that was initially

described for S. cerevisiae [6,18,19]. More precisely, the sav1 mutant

of C. neoformans had defective protein secretion and accumulated

exocytic vesicles at the septum and the bud during cell division.

Remarkably, these vesicles also contained a polysaccharide

destined to the extracellular space, suggesting that post-Golgi

secretion is involved with the transfer of macromolecules through

the cell wall. These findings were further supported by an

independent study showing that exposure of yeast cells to brefeldin

A, which interferes with the retrograde protein transport from the

Golgi apparatus to the endoplasmic reticulum, results in the

inhibition of polysaccharide assembly at the outer layer of the C.

neoformans cell wall [20]. In agreement with these observations, a C.

neoformans RNAi mutant strain lacking expression of Sec6p, an

88 kDa subunit of the exocytic complex that mediates polarized

targeting of secretory vesicles to active sites of exocytosis, was

unable to produce extracellular vesicles [16].

Although the studies mentioned above suggested the involvement

of conventional secretory mechanisms in the vesicular export of

polysaccharides in C. neoformans, it remained unclear whether

extracellular polysaccharides were targeted to the cell surface

exclusively in post-Golgi vesicles or via recycling endosomes [17].

Hence, the possibility that that the post-Golgi polysaccharide-

containing vesicles are sorted to a compartment other than the

plasma membrane, such as the late endosomes and the MVBs cannot

be ruled out. In fact, endosomes and MVBs can be connected to the

trans-Golgi secretory pathway [3], thus both pathways could be

involved in vesicular polysaccharide export in fungi. The molecular

machinery implied in MVB formation and sorting is widely known in

S. cerevisiae [21], but studies have not evaluated extracellular vesicle

transport and the connection to exosomes.

In this study, we characterized extracellular vesicles produced by

the model yeast S. cerevisiae. Based on recent suggestions that fungal

extracellular vesicles resemble exosomes but originate from Golgi-

related pathways [9,14,16], we also characterized vesicle fractions

from culture supernatants of mutants with defects in MVB formation

and post-Golgi secretion. Our results indicate that mutations in both

pathways affect vesicle composition. Nevertheless, yeast cells

mutated in the SEC4 gene, previously shown to accumulate

cytoplasmic vesicles [19], were defective in the secretion of vesicles

to the extracellular space, suggesting a key role for the Golgi-derived

secretory pathway in the trans-cell wall traffic in yeast cells.

Materials and Methods

Strains

The S. cerevisiae strains used in this study included RSY255,

RSY113, SEY6210 and BY4741 wild type (WT) cells and several

yeast secretory mutants, as subsequently described in this section

and summarized in Table 1. Strains RSY782, SF2642-1D, and

RSY954 are respectively, temperature sensitive sec1-1, sec4-2 and

bos1-1 (also known as sec32-1) mutants. Strains EEY6-2 and EEY9

correspond to mutant cells lacking expression of Vps23p and

Snf7p (also known as Vps32p), respectively. Strain GRH1delta is

defective in the expression of GRASP. WT and mutant cells were

cultivated in Sabouraud dextrose broth for 24 h at 25uC with

shaking. Based on recent literature, strains SF2642-1D (sec4-2) and

EEY9 (snf7) were used as prototypes of yeast cells defective in post-

Golgi or endosome-dependent vesicular secretion, respectively

[17,22]. Therefore, except for proteomic analyses where all strains

listed above were used, experiments in this study were performed

using sec4 and snf7 mutants and related WT strains.

Vesicle isolation

Extracellular vesicles from strains SEY6210 and BY4741 and

corresponding vps and grasp mutants were isolated from culture

supernatants, using variations of methods previously described

[8,14,15]. For vesicle isolation from cultures of strains RSY255

and RSY113 and the corresponding sec mutants, supernatants

were removed from 24 h cultures, cells were washed three times,

and fresh medium was added for further incubation at 37uC for 1

to 18 h at which time supernatants were removed and processed

for vesicle isolation. Cell viability was similar in WT and mutant

cultures, as determined by colony forming unit counting (data not

shown). For all cultures, yeast cells and debris were removed by

sequential centrifugation at 4,000 and 15,000 g (15 min, 4uC)

[8,14,15]. Supernatants were collected and concentrated by

approximately 20-fold using an Amicon ultrafiltration system

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11113

(cutoff = 100 kDa). The concentrate was again centrifuged at

4,000 and 15,000 g (15 min, 4uC) and passed through filtering

membranes (0.8

mm pores). Filtered fractions were finally

centrifuged at 100,000 g for 1 h at 4uC. Pellets were then washed

by three sequential suspension and centrifugation steps, each

consisting of 100,000 g for 1 h at 4uC with 0.1 M Tris buffered

saline (TBS, pH 7.4). The resulting pellets were then suspended in

fixative solution for electron microscopy analysis or prepared for

lipid and protein determinations, as described below. To avoid the

possibility of artifactual isolation of intracellular organelles from

dead cells, similar protocols were used with suspensions of heat-

killed cells instead of living yeast as described previously [15].

Vesicle-like structures were not observed under these conditions.

Transmission electron microscopy (TEM)

Pellets obtained after centrifugation of cell-free supernatants at

100,000 g were fixed with 2% glutaraldehyde in 0.1 M cacodylate

at room temperature for 2 h, and then incubated overnight in 4%

formaldehyde, 1% glutaraldehyde, and 0.1% PBS. The samples

were incubated for 90 min in 2% osmium, serially dehydrated in

ethanol, and embedded in Spurrs epoxy resin. Thin sections were

obtained on a Reichart Ultracut UCT and stained with 0.5%

uranyl acetate and 0.5% lead citrate. Samples were observed in a

JEOL 1200EX transmission electron microscope operating at

80 kV.

Light scattering analysis of vesicles

Vesicles undergo Brownian motion that translates into light

scattering fluctuations in a liquid phase. This property can be

measured by dynamic light scattering (DLS) techniques providing

information on the size and heterogeneity of the sample [11].

Measurement of vesicle size by DLS was performed in a 90Plus/BI-

MAS Multi Angle Particle Sizing analyzer (Brookhaven Instruments)

as described recently by Einsenman and colleagues [11].

Sterol analysis

Sterols are structural components of fungal extracellular vesicles

and markers of vesicle membranes [15]. For this reason, these

molecules were used in our model as molecular markers of

vesicular secretion in indirect quantification of vesicle fractions.

Sterols were also analyzed in cellular fractions, for comparative

analyses in the different strains used in this study. Intact cells were

collected by centrifugation and washed with PBS. These pellets or

vesicle fractions were first suspended in methanol and then two

volumes of chloroform were added. The mixture was vigorously

vortexed and centrifuged to discard precipitates, dried by vacuum

centrifugation and partitioned according to [23]. The lower phase,

containing neutral lipids, was recovered for analysis by high

performance thin layer chromatography (HPTLC). The lipid

extract was loaded into HPTLC silica plates (Si 60F254s,

LiChrospher, Merck, Germany) and separated using a solvent

system containing hexane:ether:acetic acid (80:40:2, v/v/v). The

plate was sprayed with a solution of 50 mg ferric chloride (FeCl

)

in a mixture of 90 ml water, 5 ml acetic acid and 5 ml sulfuric

acid. After heating at 100uC for 3–5 min, the sterol spots were

identified by the appearance of a red-violet color. Stained HPTLC

plates were digitalized using Adobe Photoshop CS (version 8.0)

and densitometrically analyzed with the Scion Image software

(version Alpha 4.0.3.2). Sterol content was also evaluated using the

quantitative fluorimetric kit ‘‘Amplex Red Sterol Assay Kit’’

(Molecular Probes). Sensitivity of sterol detection in this test is

approximately 8 ng in intact membranes, with no requirement of

lipid extraction. Vesicle pellets were suspended in 500

ml PBS and

10% of the sample was evaluated in this assay according to

manufacturer’s instructions. In all analyses, sterol content in each

fraction was normalized to the number of viable cells in yeast

cultures.

Protein identification by liquid chromatography-tandem

mass spectrometry (LC-MS/MS)

Vesicle proteomics followed the methodology recently estab-

lished for the analysis of extracellular vesicle fractions from fungal

cells [8,14]. Briefly, purified vesicles were suspended in 40

400 mM NH

HCO

, containing 8 M urea, and the disulfide

bonds were reduced by the addition of 10

ml 50 mM dithiotreitol,

followed by incubation for 15 min at 50uC. Cysteine residues were

alkylated by the addition of 100 mM iodoacetamide (10

ml),

followed by incubation for 15 min at room temperature under

protection from the light. The final concentration of urea was then

adjusted to 1 M and the mixture was supplemented with 4

sequencing-grade trypsin (Promega) for overnight digestion at

37uC. Resulting tryptic peptides were desalted in C18 reverse-

phase in-house ziptip columns (POROS R2 50, Applied

Biosystems), as described by Jurado et al. [24]. Samples were

finally redissolved in 5% acetonitrile (ACN), containing 0.5%

formic acid (FA), and loaded onto a C18-trap column. The

separation was performed on a capillary reverse-phase column

connected to a nanoHPLC system (nanoLC 1D Plus, Eksigent).

Peptides were eluted in a linear gradient (5–40%) of solvent B

(solvent A: 5% ACN/0.1% FA; solvent B: 80% ACN/0.1% FA)

during 200 min and directly analyzed in an electrospray-linear ion

trap-mass spectrometer (LTQ XL/ETD, Thermo Fisher)

equipped at the front end with a Triversa NanoMate nanospray

source (Advion). The nanospray was set at 1.35 kV and 0.2 psi N

Table 1. Yeast strains.

WT strain Mutation (mutant strain) Cellular event affected by mutation Origin Reference

RSY255 sec1-1 (RSY782) Membrane fusion* Schekman laboratory [63]

RSY113 sec4-2 (SF2642-1D) Vesicle targeting to the cell surface* Schekman laboratory [32]

RSY255 bos1-1, also known as sec32.2

(RSY954)

Vesicle targeting to the Golgi complex* Schekman laboratory [47]

SEY6210 snf7, also known as vps32 (EEY9) Vesicle invagination within multivesicular bodies Emr laboratory [22,31]

SEY6210 vps23 (EEY6-2) Late endosomal trafficking Emr laboratory [46]

BY4741 grh1, also known as grasp

(GRH1delta)

Unconventional secretion of acyl coenzyme A–binding

protein

EUROSCARF/Malhotra laboratory [39]

(*) Phenotype observed under restrictive temperature.

doi:10.1371/journal.pone.0011113.t001

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11113

pressure using a chip A (Advion). MS spectra were collected in

centroid mode at the 400–1700 m/z range and the ten most

abundant ions were subjected twice to collision induced dissoci-

ation (CID) with 35% normalized collision energy, before being

dynamically excluded for 60 sec.

All MS/MS spectra from peptides with 800–3500 Da, more

than 10 counts, and at least 15 fragments were converted into

DTA files using Bioworks v.3.3.1 (Thermo Fisher). DTA files were

submitted to database search using TurboSequest [25] and the S.

cerevisiae protein database (downloaded on June 8

, 2008 from

www.yeastgenome.org) combined with human keratin and porcine

trypsin sequences (downloaded on June 8

, 2008 from www.ncbi.

nlm.nih.gov/protein). All the sequences were used in the correct

and reverse orientations, forming a database with 13,760 entries.

The database search parameters included: i) trypsin cleavage in

both peptide termini with one missed cleavage site allowed; ii)

carbamidomethylation of cysteine residues as a fixed modification;

iii) oxidation of methionine residues as a variable modification;

and iv) 2.0 Da and 1.0 Da for peptide and fragment mass

tolerance, respectively. TurboSequest outputs were filtered with

DCn $0.05, peptide probability #0.05, and Xcorr $1.5, 2.0, and

2.5 for singly-, doubly-, and triply-charged peptides, respectively.

After filtering, the files were exported into XML formats and the

peptides sequences were assembled into proteins using an in-house

written script (Nakayasu, Sobreira, and Almeida, unpublished

data). The protein hits were re-filtered with sum of peptide Xcorr

$3.5. The false-discovery rate (FDR) was estimated as described

previously [8,14]. Proteins with shared peptides were assembled

into groups to assess the redundancy issue. For semi-quantitative

calculations, another in-house script was elaborated to combine

respective peptides and spectral counts into their respective protein

groups (Nakayasu, Sobreira, and Almeida, unpublished data).

Semi-quantitative determinations of protein abundance in each

sample were based on the calculation of the

exponentially modified

protein abundance index (emPAI), according to the methodology

proposed by Ishihama et al. [26]. emPAI Data were further

validated by the methodology described by Liu et al. [27], using

the number of spectra acquired for each protein (spectral count).

The relevant differences in protein abundance were selected when

a two-fold incremental change variation (WT vs. mutant proteins)

after spectral count and emPAI calculation was observed. A total

of 400 proteins were identified; however, those (n = 273) with less

than 10 spectra after MS/MS analysis of peptides were excluded

from the spectral count analysis. Using this approach we could

semi-quantify 127 proteins. This group of proteins was used in the

analyses detailed in Tables S1 and S2 and related data. The whole

set of results is available in Tables S3, S4 and S5.

Bioinformatics

Venn diagrams were prepared using the Venny tool, available

at http://bioinfogp.cnb.csic.es/tools/venny/index.html. Func-

tional networks in vesicle proteins were prepared using the Osprey

Network Visualization software (version 1.2.0), with the S. cerevisiae

database (available from the software). Analysis of the putative

presence of glycosylphosphatidylinositol (GPI)-anchored sequences

in vesicle proteins was performed as described recently [28], using

the GPI-anchored protein prediction program FragAnchor

(http://navet.ics.hawaii.edu/,fraganchor/NNHMM/NNHMM.

html) [29]. Correlation graphs and R

calculations were performed

with GraphPad Prism version 5.00 for Windows, GraphPad

Software, San Diego California USA, www.graphpad.com. The

presence of signal peptide cleavage sites in amino acid sequences

from vesicle proteins was predicted with the SignalP 3.0 Server

(http://www.cbs.dtu.dk/services/SignalP/), as previously de-

scribed [30].

Results

Morphological features and diameter distribution of S.

cerevisiae vesicles

Vesicle morphology was analyzed in both WT and mutant S.

cerevisiae cells. The selection of mutant strains for this analysis was

based on the hypothesis that fungal extracellular vesicles derive

from MVB-related pathways for exosome formation or from post-

Golgi conventional secretion [9,14,16,17]. In this context, our

analyses included the snf7 mutant, which shows defective MVB

formation [31], and the sec4-2 mutant, due to the implication of

the SEC4 gene in the secretion of Golgi-derived vesicles in yeast

cells [17,32].

Morphological analysis of yeast extracellular vesicles by TEM

revealed bilayered structures with varying levels of electron density

(Figure 1A). The vesicles were generally round or ovoid and

sometimes resembled multivesicular structures. No morphologic

alterations were apparent in vesicles obtained from sec4-2 and snf7

mutants. Analysis of the effective diameter of vesicle fractions

obtained from WT cells revealed average values ranging from

133.964.2 (strain SEY6210, parental of snf7) to 183.9624.0

(strain RSY113, parental of sec4-2) nm (Figure 1B). In interpreting

these numbers, and the results below, it is noteworthy that

dynamic light scattering tends to overestimate vesicle diameter

relative to other techniques (for discussion on this effect see [11]).

However, for the purposes of this study we have used dimensions

obtained for dynamic light scattering for all comparisons thus

controlling for any systematic trends in vesicle size inherent to the

technique. Vesicles from strain SEY6210 were clearly distributed

in two different populations, ranging from 50 to 75 and 180 to

250 nm in effective diameter. Mutation in the SNF7 gene had little

or no influence in vesicle diameter, which shifted from

133.964.2 nm in the vesicle fraction from WT cells to

143.562.1 nm for vesicles from the mutant cells. The two-

population profile of distribution of vesicle diameter in fractions

from the snf7 mutant was very similar to that found in vesicles from

the parental strain SEY6210. On the other hand, mutation in the

SEC4 gene significantly affected the diameter of extracellular

vesicles. While most of the vesicles from WT cells (strain RSY113)

were in the range of 100 to 200 nm, sec4-2 vesicles were

distributed in two populations. The mutant vesicles were

distributed in either a population ranging from 80 to 120 nm or

in a population with very high diameters (400–550 nm). Average

values shifted from 183.9624.0 nm (RSY113 strain) to

294.56117.9 nm (sec4-2 mutant). Hence, a SEC4 mutation

resulted in a qualitative difference in the vesicles produced by S.

cerevisiae.

Proteomic analysis reveals a complex composition in S.

cerevisiae extracellular vesicles

Previous studies with C. neoformans and H. capsulatum revealed

that fungal extracellular vesicles carry proteins with highly diverse

functions [8,14]. Many of these proteins were previously reported

to also be components of mammalian exosomes [33,34,35]. In our

model, proteomic analysis was performed with vesicles obtained

from three different WT S. cerevisiae cells, including strains

RSY113, RSY225, and SEY6210. Four hundred proteins were

identified in our analysis, with a FDR of ,1.6% in protein level.

Within this group, those with more than 10 spectra after MS/MS

analysis of peptides were used for the analyses shown in Figures 2,

3, 4, and 5 and Tables S1 and S2.

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11113

A summary of the proteins found in these three strains is

summarized in Table S1 and their detailed characterization is

shown in Tables S3 and S4.

The S. cerevisiae vesicle proteome had many similarities to the

protein profiles observed for C. neoformans and H. capsulatum [8,14].

Proteins related to diverse metabolic processes consisted of the

most abundant functional class in the S. cerevisiae vesicles, as

previously described for similar models (Figure 2 and Table S1)

[8,14]. Other classes found in the vesicles included cell

organization and biogenesis, transporters, carbohydrate metabo-

lism, stress response, protein biosynthesis, protein degradation,

protein transport and sporulation. A few molecules with unknown

function were also identified (Figure 2A). Of note, twenty four

different proteins required for cell wall modeling were identified,

including seven different glucanases and three glucanosyl trans-

ferases. Eight different heat shock proteins and other stress-related

proteins were also found in the vesicles, as well as peptidases,

vacuolar and secretory proteins. Although the protein composition

of the S. cerevisiae extracellular vesicles was multifaceted in many

aspects, most of the proteins identified in these preparations (90%)

are putatively functionally connected with one or more molecules

within the vesicular proteome (Figure 2B). The proportion of

proteins showing a high probability of bearing GPI-anchored was

approximately 4% of the total vesicular proteome, a value 4-fold

higher than those predicted for the cellular S. cerevisiae proteome

[29]. In addition, prediction of the presence of signal peptide

cleavage sites revealed that vesicle fractions also concentrated

proteins targeted to the endoplasmic reticulum and the secretory

pathway (Figure 3).

Profiles of protein composition of the S. cerevisiae vesicles were

very similar in different WT cells, as inferred from the high

percentage of vesicular proteins shared by distinct strains

(Figure 4). The variation range (5–25%) in protein identification

observed between different runs of the same sample is within the

acceptable range previously reported [36,37]. Analysis of vesicles

obtained from strains RSY113 and RSY225, which were both

cultivated at 37uC, revealed that more than 90% of the proteins

identified were common to both preparations. These values were

also high even when these strains were compared with SEY6210

cells, which were cultivated at room temperature. Proteomic

Figure 1. Morphology and diameter of

S. cerevisiae

extracellular vesicles. A. TEM of vesicles isolated from WT (WT) and mutant cells. Each

individual panel exemplifies the typical vesicle morphology for the cell group specified in the top. WT fractions shown in left panels were obtained

from strain RSY113, parental of the sec4-2 mutant. Similar morphological features (not shown) were observed in vesicle fractions obtained from strain

SEY6210 (parental of the snf7 mutant). Scale bar, 100 nm. B. Light scattering analysis showing diameter distribution and average values of vesicles

obtained from WT (WT) or mutant (sec4-2 and snf7) cells.

doi:10.1371/journal.pone.0011113.g001

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11113

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11113

analysis was also performed in vesicle fractions from S. cerevisiae

secretion mutants (Table S3). Remarkably, most of the proteins

found in WT cells were detected in the mutant fractions. The

qualitative analysis shown in Figure 4 revealed that most of the

proteins found in WT cells were also present in both sec4-2 and

snf7 mutants, suggesting that the protein compositions were not

severely affected by mutations in the secretion genes studied here.

Aiming at a more reliable data interpretation, we included in this

analysis vesicle proteomics of additional secretion-related systems,

which comprised mutations in SEC1, BOS1 (post-Golgi secretion)

and VPS23 (MVB formation) genes. The results obtained were

very similar to those detailed in Figure 4 (data not shown).

Protein abundance in S. cerevisiae vesicles is affected by

mutations in SEC and MVB-related genes

Based on the fact that vesicle protein composition was barely

affected by mutations in SEC4, SEC1, BOS1, VPS23, and SNF7

genes, we evaluated whether the relative abundance of vesicle

proteins would be modified in cells bearing defects in expression of

the related proteins. These analyses were based on semi-

quantitative determinations of the abundance of each vesicle

protein [26,27], the same approach used before to describe the

proteomic composition of different compartments of the secretory

pathway [38]. These methods are based on the fact that the most

abundant proteins have a higher coverage by LC-MS/MS. For

these analyses we only considered the 127 proteins with at least 10

spectra on one of the samples (Table S5). Within this group,

seventy three proteins (57.5%) had changes in their abundance in

at least one of the yeast mutants relative to the wild type strain. In

the SEC mutant group, the abundance of 24 vesicular proteins

increased, whereas 11 decreased. Analysis of the VPS group of

mutants revealed that vesicular protein abundance increased in 14

proteins and decreased in 33. Notably, the collective analysis of

graphs correlating emPAI values in vesicle proteins from WT cells

with those from mutant strains (Figure 5) demonstrated that the

most expressive changes in protein abundance were observed in

the snf7 mutant, followed by sec4-2, sec1-1, bos1-1 and vps23 cells.

Individual analysis of protein abundance revealed that the greatest

changes (5-fold increase or decrease in comparison to protein

abundance in fractions from WT cells) were observed in the VPS

gene family (Table S5). This analysis revealed an increased

abundance of plasma membrane H+-ATPases and mannosyl-

transferases in the vps mutants, with a parallel decrease of

glucanases and cyclophilin. These results, which were essentially

confirmed by spectral count (data not shown), suggest that vesicle

formation is probably affected by multiple elements of the

secretory apparatus.

Mutations in genes involved in either conventional or

unconventional secretion affect S. cerevisiae extracellular

vesicles

The differences in protein composition led us to consider the

possibility that vesicle secretion was somehow altered in the S.

cerevisiae mutants. Due to the well known characteristic of

intracellular vesicle accumulation in the sec4-2 mutant of S.

cerevisiae and in the sav1 mutant of C. neoformans [17,19,32], we first

quantified vesicle release in these cells and in the snf7 mutant by

measuring the content of sterols in cellular and vesicle fractions by

different methods. Fluorimetric analysis (Figure 6A) revealed that

the sterol content in vesicle fractions from strains SEY6210 and

snf7 were very similar, whereas a large decrease in sterol detection

was observed in the mutant sec4-2, in comparison to its parental

strain (RSY113). These results were essentially confirmed by

chromatographic analysis (Figure 6B–C), which showed that, in

comparison to WT cells, the sec4-2 mutant showed intracellular

accumulation of sterols and reduced detection of extracellular

vesicular sterols. Similar results were observed for the sec1.1

mutant (data not shown). The distribution of vesicular and cellular

sterols was apparently not significantly affected by mutation in the

SNF7 gene.

The decreased vesicle production in the sec4-2 mutant led us to

analyze the kinetics of vesicle release by these cells, in comparison

to the WT strain. While the peak of vesicle secretion in the WT

strain occurred after 18 h, detection of sterols in vesicle fractions

from the mutant strain stagnated after 3 h (Figure 6D). This result

confirmed that release of vesicles to the extracellular space was

affected by mutation in the SEC4 gene.

Due to the clear involvement of elements of the post-Golgi

secretory pathway in the kinetics of vesicle release in yeast and the

potential involvement of MVB-related pathways with vesicle

composition, we asked whether other secretion pathways could

be related to vesicular secretion in S. cerevisiae. Extracellular vesicle

fractions from WT cells and a mutant lacking expression of

GRASP, which participates in secretory mechanisms connecting

Figure 2. Functional distribution of proteins in extracellular vesicle fractions obtained from

S. cerevisiae

WT cells. A. Proteins were

grouped by color as indicated according to their function in the cellular metabolism. B. Functional interrelationship in the collection of vesicular

proteins. For protein identification according with each individual code, see Table S1.

doi:10.1371/journal.pone.0011113.g002

Figure 3. Prediction of the presence of GPI-anchored sequenc-

es and signal peptide cleavage sites in

S. cerevisiae

cellular or

vesicle proteins. All values used in these analysis were obtained in

this study, except those related to GPI-anchored sequences in cellular

fractions (y) [29].

doi:10.1371/journal.pone.0011113.g003

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11113

early endosomal compartments and the MVB sorting pathway

[39], were analyzed. As observed for the sec4.2 mutant, yeast cells

lacking GRASP expression had significantly reduced sterol content

in vesicle fractions and increased intracellular sterol accumulation

(Figure 7). This result, which suggests the participation of an

unconventional secretion pathway in vesicle production, is in

agreement with the fact that approximately 75% of vesicular

proteins have no signal peptide sequences for ER translation

(Figure 3).

Discussion

The production of extracellular vesicles by yeast cells has now

been reported for at least 6 different fungal species

[8,10,11,13,14,15,16]. Fungal extracellular vesicles are believed

to function as carriers of distinct molecules to the extracellular

space which, in the case of pathogens, includes a wide range of

virulence factors, including polysaccharides, pigments, and lipids

[8,10,11,13,14]. As described for mammalian exosomes [40], the

vesicular transport in fungi may even include the release of nucleic

acids to the extracellular milieu [41]. The secretion of such a

complex array of different molecules is expected to impact the

interaction of fungal cells with their hosts. In fact, extracellular

vesicles isolated from the yeast pathogen C. neoformans were

recently reported to modulate macrophage functions [42].

Vesicular traffic in eukaryotes is a complex and multifunctional

cellular mechanism. Intracellular vesicles are required for the

traffic of proteins destined for secretion, in pathways that require

their movement from the endoplasmic reticulum to the Golgi

complex and then, via the trans-Golgi network, to the cell surface

[5,19,43]. These vesicles are expected to fuse with the plasma

membrane releasing their cargo. Vesicular secretion can also

involve exosomes, which originate from intracellular MVBs. These

organelles derive from endosomes, which form internal vesicles

that are released to the extracellular milieu as exosomes upon

plasma fusion with the plasma membrane [3]. Yeast extracellular

vesicles have been generally termed ‘fungal exosomes’ [9,14,16],

but different studies suggest that they are linked to elements of the

conventional post-Golgi secretory pathway [16,17,20]. It remains

unknown, nevertheless, whether the formation of MVBs is related

to the biogenesis of fungal extracellular vesicles.

C. neoformans represents the yeast cell model that extracellular

vesicles have been investigated in most detail. This species appears

to use trans-cell wall vesicular transport to release its major

capsular polysaccharide [15,16,17,20], a high molecular weight

polysaccharide known as glucuronoxylomannan (GXM). Although

it has been suggested that C. neoformans produces MVB- and

exosome-like structures [14,44,45], the vesicular traffic of GXM

apparently requires homologues of SEC4 and SEC6 genes [16,17],

suggesting that the export of polysaccharide-containing vesicles in

these cells requires events of the conventional post-Golgi secretory

apparatus. The events required for the biogenesis of extracellular

vesicles in these cells are unclear, but the complex and variable

morphology of extracellular cryptococcal vesicles analyzed by

TEM [14] strongly suggests that the fractions usually analyzed in

studies on fungal extracellular vesicles include mixed populations

of diverse cellular origin.

Although C. neoformans was the species that led to discovery of

extracellular vesicles, this fungus may not be the ideal system at

this time for genetic dissection of vesicular physiology because the

copious extracellular polysaccharide hinders several analytic

approaches such as mass spectrometry and genetic tools remain

more difficult to use relative to other model fungi. Consequently,

we turned our attention to S. cerevisiae, where we similarly detected

extracellular vesicles in culture supernatants by TEM [8].

Consequently we took advantage of the availability of S. cerevisiae

secretion mutants and characterized their extracellular vesicles,

aiming to identify key elements required for the generation of these

compartments in yeast cells. Two major prototypes were used in

our study, based on previous literature observations. Snf7,a

mutant strain with defective MVB formation [31], was selected as

a candidate to evaluate whether exosome formation was related to

extracellular vesicles in yeast cells. The sec4-2 mutant was selected

as the prototype mutant to evaluate whether events of the post-

Golgi conventional secretion were required for the release of

fungal extracellular vesicles, given a recent report that the

orthologue of SEC4 in C. neoformans is required for the export of

polysaccharide-containing vesicles [17]. Using the same rationale,

some of the experiments performed in this study included mutant

cells with related defects in post-Golgi secretion mechanisms (sec1-

1 and bos1-1) and MVB biogenesis (vps23) [19,46,47].

As determined in this work and in a previous study [11], the

diameter of fungal extracellular vesicles ranged from 50 to

500 nm. These dimensions contrast with the fact that extracellular

vesicles in other models are in a diameter range lower than

100 nm [3]. Different studies, however, have demonstrated that

larger membrane structures (300–500 nm in size) are the vehicles

Figure 4. Venn diagrams showing similarities of protein composition in vesicle fractions from WT (RSY113 and SEY6210 strains)

and mutant cells (

sec4-2

and

snf7

mutants).

doi:10.1371/journal.pone.0011113.g004

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11113

responsible for long distance, ER-to-Golgi and trans-Golgi to

plasma membrane transport of secretory molecules (reviewed in

[48]). Estimation of the diameter of cell wall pores in yeast cells

revealed values in the range of 200 to 400 nm [49], which would

theoretically permit the release of vesicles of different sizes to the

extracellular space. These observations and the high variability in

the morphology of fungal extracellular vesicles [14] support the

hypothesis that the vesicle populations originate from compart-

ments of distinct biogenesis, which could involve both Golgi- and

exosome-derived pathways.

Proteomic analysis of yeast extracellular vesicles revealed a

complex composition, as described for mammalian exosomes and

other fungal vesicles [14,33,34,35]. Several cytoplasmic proteins

with no apparent relation with secretory processes were observed

in the vesicle proteome, providing another parallel with mamma-

lian exosomes. Sorting of cytosolic proteins into exosomes is

normally explained by a random engulfment of small portions of

cytosol during the inward budding process of MVBs [3]. Of note,

many cell wall-degrading enzymes were found in the S. cerevisiae

vesicles, consistent with a prior notion that the passage of vesicles

through the cell wall could require hydrolysis of structural

components [8,9,12]. These enzymes were present in all fractions

analyzed in this study. Protein composition was consistently similar

in all vesicle fractions analyzed, providing confidence in the

Figure 5. Correlation analysis of the relative variation of protein abundance in vesicle fractions from WT (WT) or mutant

S. cerevisiae

cells. emPAI values for each parental strain were plotted against the values obtained for yeast mutant fractions analyzed by proteomics. SEC or MVB-

related mutants included snf7 (A), sec4-2 (B), vps23 (C), sec1-1 (D) and bos1-1 (E). Lower R

values suggest greater alterations in relative protein

distribution in vesicles from the mutants, in comparison to WT cells. Proteins whose abundance was increased in vesicle fractions from mutant cells

are represented by the red spots, whereas proteins that were more abundant in WT fractions are highlighted in blue.

doi:10.1371/journal.pone.0011113.g005

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11113

validity of the conclusion that vesicle proteins include many

different functional classes.

Most of the proteins found in the yeast vesicle proteome were

potentially associated other molecules identified in the vesicular

protein collection. More precisely, bioinformatics analyses sug-

gested that at least 219 protein-protein interactions were observed

within the vesicle proteome. Some of these interactions were in

fact expected. For example, YGR032W and YLR342W (glucan

synthases) were associated, and they are in the same functional

class. Apparently unrelated proteins, however, were also poten-

tially interacting. For instance, YER103W is a heat shock protein

that plays a role in protein-membrane targeting and translocation.

We found that the molecule interacted with other vesicular heat

shock proteins, but also with the metabolic enzyme glyceralde-

hyde-3-phosphate dehydrogenase [50,51,52]. Similarly,

YLR249W is a translational elongation factor that interacted with

four other elongation factors and two heat shock proteins, but also

with pyruvate decarboxylase, phosphoglucose isomerase and

alcohol dehydrogenase [53,54,55,56]. These observations illustrate

the complexity of the protein composition of fungal extracellular

vesicles, as well as the difficulties in unraveling their biosynthetic

steps.

Vesicular fractions had greater concentrations of secretory and

GPI-anchored proteins in comparison to intact S. cerevisiae cells.

Figure 6. Sterol analysis in vesicle and cellular fractions obtained from yeast cells. A–B. Indirect sterol-based vesicle quantification of

fractions obtained from cultures of WT (WT) or mutant cells. The sterol content was determined by fluorimetric methods (A) or by densitometric

analysis of bands obtained after HPTLC separation (B). Comparative analysis of the sterol content in vesicle fractions obtained from WT or mutant

cells suggested that the sec4-2 mutant has a defective release of extracellular vesicles. This supposition was supported by chromatographic analysis in

association with densitometry of cellular (C) or vesicle (D) fractions obtained from yeast cultures. The sec4-2 mutant, in contrast to the snf7 mutant,

showed intracellular accumulation of sterols (C) and a lower kinetics of release of sterol-containing vesicles (D). Arrows indicate the migration ofan

ergosterol standard in TLC plates. Results are representative of three independent analyses showing similar results.

doi:10.1371/journal.pone.0011113.g006

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11113

This observation suggests that the unconventional mechanism of

protein secretion by vesicle release includes elements of the

conventional secretory pathway. Furthermore, the fact that

sequences potentially containing GPI anchors and signals for ER

targeting are enriched in vesicle fractions confirms previous

observations that fungal extracellular vesicles are actively secreted

rather than released by dead cells, since they concentrate

secretion-related proteins.

Our semi-quantitative analysis of protein abundance in yeast

vesicles strongly suggests that fungal vesicles include compart-

ments related to the MVB-derived pathway of exosome

formation. Although vesicle release was similar in WT cells and

in the snf7 mutant, the abundance of 35 proteins was modified in

the mutant vesicles, suggesting that defects in MVB formation

also affect extracellular vesicles in fungi. In fact, the greatest

differences in protein abundance in WT/mutant systems

observed in this study involved vps mutants and, particularly,

snf7 cells. In vesicle fractions from these mutants, proteins with

the greatest levels of increase in relative abundance consisted of

two related plasma membrane proton ATPases (YGL008C and

YPL036W; Pma1p and Pma2p, respectively) and two Golgi

mannosyltransferases (YDR483W and YBR199W). The manno-

syltransferases YDR483W and YBR199W are known to interact

with other Golgi proteins, which include, respectively, members

of the exocyst complex and t-SNAREs required for vesicular

transport [57,58]. Similarly, Pma1p and its isoform Pma2p

interact with several elements of Golgi-associated pathways of

cellular traffic, including Ric1p, a protein involved in retrograde

transport to the cis-Golgi network, and Vps29p, which is essential

for endosome-to-Golgi retrograde transport [59,60]. Therefore,

we speculate that mutations in the VPS genes could led to the

activation of compensatory mechanisms of Golgi-associated

traffic, which could explain the increased abundance of Golgi-

related proteins in vesicles from snf7 and vps2 3 cells. On the other

hand, vesicles from the snf7 mutants had significantly decreased

levels of a protein of unknown function, two glucanases and

cyclophilin. The functional implication of each of these individual

changes in protein abundance is unknown, but the possibilities

are numerous. For instance, cyclophilin is supposed to interact

with 34 different proteins in S. cerevisiae, including elements of the

secretory apparatus [60] and cell wall architecture [61]. These

observations illustrate the fact that vesicular proteins with no

apparent connections with the secretory process may be directly

or indirectly linked to vesicle biogenesis.

Our results show that mutation of the SEC4 gene is associated

with a delay in vesicle release to the extracellular space. This result

is supportive and consistent with previous reports that a sec4-2

mutant of S. cerevisiae and a similar mutant in C. neoformans

accumulate intracellular vesicles [17,18]. This observation sup-

ports the view that the extracellular vesicles observed in fungal

cells may not be conventional exosomes, as suggested in

independent studies [14,16,17]. Nevertheless, it remains unknown

how post-Golgi vesicles, which are expected to fuse with the

plasma membrane to release their cargo, would leave the cell wall.

Different reports, however, suggest that not all secretory vesicles

fuse with the plasma membrane (reviewed in [48]), which supports

a prior study with a sec6 C. neoformans mutant [16] and our current

observations with S. cerevisiae strains showing that post-Golgi

secretion events are required for the release of extracellular

vesicles.

Remarkably, vesicle release was not completely abrogated in

any of the mutants analyzed in this study. This observation could

indicate that multiple cellular pathways are required for formation

of fungal extracellular vesicles, including elements of non-

conventional secretory mechanisms. In fact, in our study, mutant

cells lacking expression of GRASP, which is required for

unconventional secretion of an acyl coenzyme A–binding protein

in S. cerevisiae [39] and Dictyostelium discoideum [4], showed a

decreased content of extracellular vesicles, in comparison with WT

cells. This observation may be related to the fact that acyl

coenzyme A–binding protein plays an important role in the

cellular distribution of sphingolipids [62], which are important

structural components of fungal extracellular vesicles [15].

GRASP is involved in secretory mechanisms that require

autophagy genes, early endosomal compartments, and MVBs

[39]. The decreased vesicular release by the GRASP mutant

reinforces the importance of Golgi components in extracellular

vesicle formation and supports the notion that extracellular release

of vesicles in fungi is a multifactorial cellular event of high

complexity, and possibly involves considerable redundancy. We

cannot rule out the possibility, however, that the collection of

mutations analyzed in our study are affecting different types of

vesicles, since the methods currently used for vesicle purification

do not discriminate between vesicles of different origins, resulting

in heterogeneous preparations.

Our current results together with previous reports suggest

agreement with the fact that endosomes and MVBs can be

connected to the trans-Golgi secretory pathway [3], which could

directly affect the formation of fungal extracellular vesicles. In

summary, after analysis of eight different S. cerevisiae strains, our

results indicate that both MVB- and Golgi-derived cellular

pathways affect the formation and release of extracellular vesicles

Figure 7. Sterol analysis in vesicle (A) and cellular (B) fractions obtained from WT cells and a mutant strain lacking GRASP

expression. Comparative analysis of the sterol content in vesicle fractions obtained from WT or mutant cells suggested that GRASP is involved in the

release of extracellular vesicles. Chromatograms and related densitometric analyses are shown. Arrows indicate the chromatographic migration of

ergosterol standards. Results are representative of three independent analyses showing similar results.

doi:10.1371/journal.pone.0011113.g007

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11113

by fungal cells. We believe our observations with S. cerevisiae

extracellular vesicles will contribute to the understanding of a

complex event in the biology of yeast cells. Since yeast

extracellular vesicles in pathogens are presumably linked to fungal

virulence and the ability of fungal cells to modulate the host

immunity, these results could also be of use in the design of

pathogenic models aiming at the elucidation of the role of

secretion events in fungal virulence.

Supporting Information

Table S1 Proteomic analysis of S. cerevisiae extracellular

vesicles*.

Found at: doi:10.1371/journal.pone.0011113.s001 (0.16 MB

DOC)

Table S2 Changes in protein abundance in vesicle fractions

from secretion mutants of S. cerevisiae, in comparison to fractions

from WT cells.

Found at: doi:10.1371/journal.pone.0011113.s002 (0.09 MB

DOC)

Table S3 All identified proteins.

Found at: doi:10.1371/journal.pone.0011113.s003 (0.18 MB

XLS)

Table S4 Detailed information of peptide identification.

Found at: doi:10.1371/journal.pone.0011113.s004 (0.72 MB

XLS)

Table S5 emPAI calculation and spectral count for the 128

proteins used for the semi-quantitative analysis.

Found at: doi:10.1371/journal.pone.0011113.s005 (0.08 MB

XLS)

Acknowledgments

DLO is a PhD student at Instituto de Bioquı

mica Me´dica (Federal

University of Rio de Janeiro, Brazil). We thank the Biomolecule Analysis

Core Facility, Border Biomedical Research Center/Biology/University of

Texas at El-Paso for the access to the mass spectrometry instrumentation.

We are also grateful to Jorge J. Jo´ Bastos Ferreira for helpful comments and

Guylaine Poisson for help with use of Fraganchor. We are particularly

thankful to Drs. Randy Schekman, Scott Emr, Vivek Malhotra and Juan

Duran for donation of yeast mutants and helpful suggestions to this study.

We are particularly grateful to Dr. Vivek Malhotra for his initial suggestion

on the role of GRASP in the release of extracellular vesicles in yeast cells.

Author Contributions

Conceived and designed the experiments: DLO ESN JDN AC ICA LN

MLR. Performed the experiments: DLO ESN LSJ AJG RJBC SF LN.

Analyzed the data: DLO ESN TJPS RJBC ICA MLR. Contributed

reagents/materials/analysis tools: JDN AC ICA MLR. Wrote the paper:

DLO JDN AC ICA LN MLR.

References

1. Glick BS, Malhotra V (1998) The curious status of the Golgi apparatus. Cell 95:

883–889.

2. Nickel W, Rabouille C (2009) Mechanisms of regulated unconventional protein

secretion. Nat Rev Mol Cell Biol 10: 148–155.

3. Keller S, Sanderson MP, Stoeck A, Altevogt P (2006) Exosomes: from biogenesis

and secretion to biological function. Immunol Lett 107: 102–108.

4. Kinseth MA, Anjard C, Fuller D, Guizzunti G, Loomis WF, et al. (2007) The

Golgi-associated protein GRASP is required for unconventional protein

secretion during development. Cell 130: 524–534.

5. Schekman R (2002) Lasker Basic Medical Research Award. SEC mutants and

the secretory apparatus. Nat Med 8: 1055–1058.

6. Schekman R, Novick P (2004) 23 genes, 23 years later. Cell 116: S13–15,

11 p following S19.

7. Wickner W, Schekman R (2005) Protein translocation across biological

membranes. Science 310: 1452–1456.

8. Albuquerque PC, Nakayasu ES, Rodrigues ML, Frases S, Casadevall A, et al.

(2008) Vesicular transport in Histoplasma capsulatum: an effective mechanism

for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol

10: 1695–1710.

9. Casadevall A, Nosanchuk JD, Williamson P, Rodrigues ML (2009) Vesicular

transport across the fungal cell wall. Trends Microbiol 17: 158–162.

10. De Jesus M, Nicola AM, Rodrigues ML, Janbon G, Casadevall A (2009)

Capsular localization of the Cryptococcus neoformans polysaccharide compo-

nent galactoxylomannan. Eukaryot Cell 8: 96–103.

11. Eisenman HC, Frases S, Nicola AM, Rodrigues ML, Casadevall A (2009)

Vesicle-associated melanization in Cryptococcus neoformans. Microbiology 155:

3860–3867.

12. Nosanchuk JD, Nimrichter L, Casadevall A, Rodrigues ML (2008) A role for

vesicular transport of macromolecules across cell walls in fungal pathogenesis.

Commun Integr Biol 1: 37–39.

13. Oliveira DL, Nimrichter L, Miranda K, Frases S, Faull KF, et al. (2009)

Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals

an intimate association between membrane lipids and glucuronoxylomannan.

Fungal Genet Biol 46: 956–963.

14. Rodrigues ML, Nakayasu ES, Oliveira DL, Nimrichter L, Nosanchuk JD, et al.

(2008) Extracellular vesicles produced by Cryptococcus neoformans contain

protein components associated with virulence. Eukaryot Cell 7: 58–67.

15. Rodrigues ML, Nimrichter L, Oliveira DL, Frases S, Miranda K, et al. (2007)

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic

solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6:

48–59.

16. Panepinto J, Komperda K, Frases S, Park YD, Djordjevic JT, et al. (2009) Sec6-

dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus

neoformans. Mol Microbiol 71: 1165–1176.

17. Yoneda A, Doering TL (2006) A eukaryotic capsular polysaccharide is

synthesized intracellularly and secreted via exocytosis. Mol Biol Cell 17:

5131–5140.

18. Walworth NC, Goud B, Kabcenell AK, Novick PJ (1989) Mutational analysis of

SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic.

EMBO J 8: 1685–1693.

19. Schekman R, Esmon B, Ferro-Novick S, Field C, Novick P (1983) Yeast

secretory mutants: isolation and characterization. Methods Enzymol 96:

802–815.

20. Hu G, Steen BR, Lian T, Sham AP, Tam N, et al. (2007) Transcriptional

regulation by protein kinase A in Cryptococcus neoformans. PLoS Pathog 3:

e42.

21. Hurley JH, Emr SD (2006) The ESCRT complexes: structure and mechanism of

a membrane-trafficking network. Annu Rev Biophys Biomol Struct 35: 277–298.

22. Babst M, Katzmann DJ, Estepa-Sabal EJ, Meerloo T, Emr SD (2002) Escrt-III:

an endosome-associated heterooligomeric protein complex required for mvb

sorting. Dev Cell 3: 271–282.

23. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation

and purification of total lipides from animal tissues. J Biol Chem 226: 497–509.

24. Jurado JD, Rael ED, Lieb CS, Nakayasu E, Hayes WK, et al. (2007)

Complement inactivating proteins and intraspecies venom variation in Crotalus

oreganus helleri. Toxicon 49: 339–350.

25. Eng JK, McCormack AL, Yates JR, 3rd (1994) An approach to correlate tandem

mass spectral data of peptides with amino acid sequences in a protein database.

J Am Soc Mass Spectrom 5: 976–989.

26. Ishihama Y, Oda Y, Tabata T, Sato T, Nagasu T, et al. (2005) Exponentially

modified protein abundance index (emPAI) for estimation of absolute protein

amount in proteomics by the number of sequenced peptides per protein. Mol

Cell Proteomics 4: 1265–1272.

27. Liu H, Sadygov RG, Yates JR, 3rd (2004) A model for random sampling and

estimation of relative protein abundance in shotgun proteomics. Anal Chem 76:

4193–4201.

28. Nakayasu ES, Yashunsky DV, Nohara LL, Torrecilhas AC, Nikolaev AV, et al.

(2009) GPIomics: global analysis of glycosylphosphatidylinositol-anchored

molecules of Trypanosoma cruzi. Mol Syst Biol 5: 261.

29. Poisson G, Chauve C, Chen X, Bergeron A (2007) FragAnchor: a large-scale

predictor of glycosylphosphatidylinositol anchors in eukaryote protein sequences

by qualitative scoring. Genomics Proteomics Bioinformatics 5: 121–130.

30. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of

signal peptides: SignalP 3.0. J Mol Biol 340: 783–795.

31. Tu J, Vallier LG, Carlson M (1993) Molecular and genetic analysis of the SNF7

gene in Saccharomyces cerevisiae. Genetics 135: 17–23.

32. Salminen A, Novick PJ (1987) A ras-like protein is required for a post-Golgi

event in yeast secretion. Cell 49: 527–538.

33. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E, et al. (2007) Identification

and characterization of microvesicles secreted by 3T3-L1 adipocytes: redox- and

hormone-dependent induction of milk fat globule-epidermal growth factor 8-

associated microvesicles. Endocrinology 148: 3850–3862.

34. Gatti JL, Metayer S, Belghazi M, Dacheux F, Dacheux JL (2005) Identification,

proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles.

Biol Reprod 72: 1452–1465.

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 12 June 2010 | Volume 5 | Issue 6 | e11113

35. Mears R, Craven RA, Hanrahan S, Totty N, Upton C, et al. (2004) Proteomic

analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel

electrophoresis and mass spectrometry. Proteomics 4: 4019–4031.

36. Schirle M, Heurtier MA, Kuster B (2003) Profiling core proteomes of human cell

lines by one-dimensional PAGE and liquid chromatography-tandem mass

spectrometry. Mol Cell Proteomics 2: 1297–1305.

37. Faca V, Pitteri SJ, Newcomb L, Glukhova V, Phanstiel D, et al. (2007)

Contribution of protein fractionation to depth of analysis of the serum and

plasma proteomes. J Proteome Res 6: 3558–3565.

38. Gilchrist A, Au CE, Hiding J, Bell AW, Fernandez-Rodriguez J, et al. (2006)

Quantitative proteomics analysis of the secretory pathway. Cell 127: 1265–1281.

39. Duran JM, Anjard C, Stefan C, Loomis WF, Malhotra V () Unconventional

secretion of Acb1 is mediated by autophagosomes. J Cell Biol 188: 527–536.

40. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, et al. (2007) Exosome-

mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic

exchange between cells. Nat Cell Biol 9: 654–659.

41. Nicola AM, Frases S, Casadevall A (2009) Lipophilic dye staining of

Cryptococcus neoformans extracellular vesicles and capsule. Eukaryot Cell 8:

1373–1380.

42. Oliveira DL, Freire-de-Lima CG, Nosanchuk JD, Casadevall A, Rodrigues ML,

et al. Extracellular vesicles from Cryptococcus neoformans modulate macro-

phage functions. Infect Immun.

43. Novick P, Field C, Schekman R (1980) Identification of 23 complementation

groups required for post-translational events in the yeast secretory pathway. Cell

21: 205–215.

44. Takeo K, Uesaka I, Uehira K, Nishiura M (1973) Fine structure of Cryptococcus

neoformans grown in vivo as observed by freeze-etching. J Bacteriol 113:

1449–1454.

45. Takeo K, Uesaka I, Uehira K, Nishiura M (1973) Fine structure of Cryptococcus

neoformans grown in vitro as observed by freeze-etching. J Bacteriol 113:

1442–1448.

46. Babst M, Odorizzi G, Estepa EJ, Emr SD (2000) Mammalian tumor

susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both

function in late endosomal trafficking. Traffic 1: 248–258.

47. Wuestehube LJ, Duden R, Eun A, Hamamoto S, Korn P, et al. (1996) New

mutants of Saccharomyces cerevisiae affected in the transport of proteins from

the endoplasmic reticulum to the Golgi complex. Genetics 142: 393–406.

48. Stephens DJ, Pepperkok R (2001) Illuminating the secretory pathway: when do

we need vesicles? J Cell Sci 114: 1053–1059.

49. de Souza Pereira R, Geibel J (1999) Direct observation of oxidative stress on the

cell wall of Saccharomyces cerevisiae strains with atomic force microscopy. Mol

Cell Biochem 201: 17–24.

50. Werner-Washburne M, Stone DE, Craig EA (1987) Complex interactions

among members of an essential subfamily of hsp70 genes in Saccharomyces

cerevisiae. Mol Cell Biol 7: 2568–2577.

51. Martineau CN, Beckerich JM, Kabani M (2007) Flo11p-independent control of

‘‘mat’’ formation by hsp70 molecular chaperones and nucleotide exchange

factors in yeast. Genetics 177: 1679–1689.

52. Gong Y, Kakihara Y, Krogan N, Greenblatt J, Emili A, et al. (2009) An atlas of

chaperone-protein interactions in Saccharomyces cerevisiae: implications to

protein folding pathways in the cell. Mol Syst Biol 5: 275.

53. Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, et al. (2006) Global landscape

of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:

637–643.

54. Collins SR, Kemmeren P, Zhao XC, Greenblatt JF, Spencer F, et al. (2007)

Toward a comprehensive atlas of the physical interactome of Saccharomyces

cerevisiae. Mol Cell Proteomics 6: 439–450.

55. Chakraburtty K, Triana-Alonso FJ (1998) Yeast elongation factor 3: structure

and function. Biol Chem 379: 831–840.

56. Kovalchuke O, Kambampati R, Pladies E, Chakraburtty K (1998) Competition

and cooperation amongst yeast elongation factors. Eur J Biochem 258: 986–993.

57. Inadome H, Noda Y, Adachi H, Yoda K (2005) Immunoisolaton of the yeast

Golgi subcompartments and characterization of a novel membrane protein,

Svp26, discovered in the Sed5-containing compartments. Mol Cell Biol 25:

7696–7710.

58. De Craene JO, Coleman J, Estrada de Martin P, Pypaert M, Anderson S, et al.

(2006) Rtn1p is involved in structuring the cortical endoplasmic reticulum. Mol

Biol Cell 17: 3009–3020.

59. Luo W, Chang A (1997) Novel genes involved in endosomal traffic in yeast

revealed by suppression of a targeting-defective plasma membrane ATPase

mutant. J Cell Biol 138: 731–746.

60. Schuldiner M, Collins SR, Thompson NJ, Denic V, Bhamidipati A, et al. (2005)

Exploration of the function and organization of the yeast early secretory

pathway through an epistatic miniarray profile. Cell 123: 507–519.

61. Costanzo M, Baryshnikova A, Bellay J, Kim Y, Spear ED, et al. The genetic

landscape of a cell. Science 327: 425–431.

62. Gaigg B, Neergaard TB, Schneiter R, Hansen JK, Faergeman NJ, et al. (2001)

Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and

causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae.

Mol Biol Cell 12: 1147–1160.

63. Novick P, Schekman R (1979) Secretion and cell-surface growth are blocked in a

temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad

Sci U S A 76: 1858–1862.

Yeast Extracellular Vesicles

PLoS ONE | www.plosone.org 13 June 2010 | Volume 5 | Issue 6 | e11113

www.landesbioscience.com Communicative & Integrative Biology 533

AUTOPHAGIC PUNCTUM

ARTICLE ADDENDUM

Addendum to: Oliveira DL, Nakayasu ES, Joe LS,

Guimaraes AJ, Sobreira TJP, Nosanchuk JD, et al.

Characterization of yeast extracellular vesicles:

evidence for the participation of dierent path-

ways of cellular trac in vesicle biogenesis. PLoS

ONE 2010; 5:11113; PMID: 20559436; DOI: 0.1371/

jo ur nal .p on e.0 011113 .

Key words: secretion, extracellular

vesicles, exosome, trans-cell wall

transport, yeast box

Submitted: 06/21/10

Accepted: 06/21/10

Previously published online:

www.landesbioscience.com/journals/cib/

article/12756

DOI: 10.4161/cib.3.6.12756

*Correspondence to: Marcio L. Rodrigues;

Email: [email protected]j.br

he cellular events required for

unconventional protein secretion in

eukaryotic pathogens are beginning to be

revealed. In fungi, extracellular release

of proteins involves passage through

the cell wall by mechanisms that are

poorly understood. In recent years, sev-

eral studies demonstrated that yeast cells

produce vesicles that traverse the cell

wall to release a wide range of cellular

components into the extracellular space.

These studies suggested that extracellu-

lar vesicle release involves components of

both conventional and unconventional

secretory pathways, although the precise

mechanisms required for this process are

still unknown. We discuss here cellular

events that are candidates for regulating

this interesting but elusive event in the

biology of yeast cells.

Protein secretion is a widely studied cel-

lular phenomenon. To reach the extracel-

lular milieu, intracellularly synthesized

proteins are targeted to the cell surface for

release to the extracellular space.

In mam-

malian cells, the plasma membrane is the

ﬁnal barrier to be crossed during secre-

tion. Such processes, which involve both

conventional and unconventional mecha-

nisms, have been studied in detail and a

number of excellent reviews are available

in the literature.

1-6

Secretory systems in microbes

and mammalian cells show points of

Biogenesis of extracellular vesicles in yeast

Many questions with few answers

Débora L. Oliveira,

Ernesto S. Nakayasu,

2,†

Luna S. Joffe,

Allan J. Guimarães,

Tiago J.P. Sobreira,

Joshua D. Nosanchuk,

3,5

Radames J.B. Cordero,

Susana Frases,

Arturo Casadevall,

3,5

Igor C. Almeida,

Leonardo Nimrichter

and Marcio L. Rodrigues

Laboratório de Estudos Integrados em Bioquímica Microbiana; Instituto de Microbiologia Professor Paulo de Góes; Rio de Janeiro; Brazil;

The Border

Biomedical Research Center; Department of Biological Sciences; University of Texas at El Paso; El Paso, TX USA; Departments of

Medicine and

Microbiology

and Immunology; Albert Einstein College of Medicine; Bronx, New York USA;

Laboratory of Genetics and Molecular Cardiology; Heart Institute (InCor);

University of Sao Paulo; São Paulo, SP Brazil;

Laboratório de Biotecnologia; Instituto Nacional de Metrologia; Normalização e Qualidade Industrial; Rio de

Janeiro, Brazil

†

Current address: Paciﬁc Northwest National Laboratory; Richland, WA USA

convergence and divergence.

Fungi and

prokaryotes are surrounded by thick cell

walls, a key difference in comparison with

mammalian and other eukaryotic cells

(e.g., protozoa) that adds signiﬁcant com-

plexity to secretion systems in these organ-

isms. A number of mechanisms have been

proposed for the trans-cell wall molecular

transport in prokaryotes.

In fungi, how-

ever, the mechanisms required for passage

of molecules across the cell wall are poorly

understood. Recently, extracellular vesicle

release has been described as a mechanism

used by yeast cells to secrete many mol-

ecules across the cell wall.

9-12

Extracellular vesicles produced by

fungal cells share morphological and bio-

chemical similarities with mammalian

exosomes,

13,14

including an ability to mod-

ulate the function of immune cells.

Plant

cells also produce exosome-like vesicles,

supporting the notion that vesicular

release is a mechanism of trans-cell wall

passage shared by cell-wall containing

eukaryotes. In contrast to what is observed

for mammalian exosomes,

the pathways

required for extracellular vesicle biogenesis

and release in both plant and fungal cells

remain virtually unknown. One remark-

able feature of mammalian exosomes and

fungal extracellular vesicles is the abun-

dance of cytoplasmic proteins lacking a

signal peptide that directs proteins to the

endoplasmic reticulum in conventional

secretory processes.

13,14,18-20

534 Communicative & Integrative Biology Volume 3 Issue 6

were observed in extracellular vesicle sam-

ples obtained from S. cerevisiae cultures.

We hypothesize that these enzymes could

hydrolyze cell wall components to facilitate

vesicle passage through this cellular barrier.

The methods currently used for vesicle

puriﬁcation do not discriminate between

vesicles of different origins. This implies

that heterogeneous preparations are

obtained during vesicle isolation. In this

context, the possibility that the collection

of mutations analyzed in our recent study

is affecting different types of vesicles can-

not be ruled out. The current knowledge on

how fungal extracellular vesicles are formed,

in fact, strongly suggests the involvement of

multiple—and perhaps still unknown—

pathways of secretion.

9,11,13

As recently

described in independent studies, uncon-

ventional protein secretion can also involve

autophagosomes,

27,2 8

which are intracellular

structures whose functions were initially

attributed to many catabolic steps.

In autophagy, cytosolic material is

sequestered by an expanding membrane

compartment, the phagophore, resulting in

the formation of a double-membrane vesi-

cle, the autophagosome.

Autophagosomes

then fuse with the lysosome/vacuole where,

as initially supposed, the sequestered mate-

rial is degraded.

Independent studies,

however, have shown that yeast cells can

also direct the autophagic content for secre-

tion, in a process called exophagy.

27-29

fact, the autophagic machinery participates

in the packaging and delivery of the soluble

yeast protein acyl-Coenzyme A-binding

protein Acb1 to the cell surface. Therefore,

these studies suggest the existence of a

vesicular mechanism that utilizes the same

machinery for both secretion and degrada-

tion of cellular components. It is interesting

to note that secretion of Acb1 from yeast as

well as secretion of the Dictyostelium discoi-

deum Acb1 homologue, AcbA, depends on

the Golgi associated protein GRASP,

27,28,30

which is apparently required for extracel-

lular vesicle release in yeast cells.

These

observations add to an already long list of

candidates that can regulate vesicle forma-

tion in yeast cells.

After our initial description of fungal

extracellular vesicles in 2007,

eight differ-

ent studies showing their functions in fun-

gal physiology or pathogenesis have been

reported in the literature.

13-15,21,22,31-33

Vesicle

their cargo, so they are not expected

to interfere with formation of vesicular

structures outside the cell.

In our analy-

ses, however, yeast mutants lacking Sec4p,

a secretory vesicle-associated Rab GTPase

essential for Golgi-derived exocytosis,

had reduced kinetics of vesicle release to

the extracellular milieu.

The fact that

cells with defects in a post-Golgi event of

secretion, but not with disturbed MVB

formation, affected vesicle release raised

an obvious and still unanswered question:

how is a double layered vesicle secreted

from yeast cells?

The simplest and more tangible expla-

nation for the release of any extracellu-

lar vesicle is the fusion of MVB with the

plasma membrane. However, studies by

our group

25,26

clearly show that double-

layered vesicles can bud from the plasma

membrane of yeast cells (Fig. 1). Therefore,

one could speculate that proteins required

for post-Golgi conventional secretion could

be required for addressing vesicle compo-

nents to the plasma membrane. Vesicles

would then be formed by membrane bud-

ding and sequential transfer to the cell wall

and extracellular space. That would be con-

sistent with previous hypotheses raising the

possibility that formation of extracellular

vesicles can involve membrane budding.

Budding from the plasma membrane would

also be in line with the complex vesicle com-

position including cytoplasmic elements, as

observed in our analyses.

13,14,21

It remains

unknown how these vesicles traverse the cell

wall, but many cell wall degrading enzymes

In a recent study, we evaluated the

contribution of both conventional and

unconventional pathways of secretion in

the formation of extracellular vesicles in

the model yeast Saccharomyces cerevisiae.

Our approach was based on the study of

mutants with defects in two major secre-

tion pathways: conventional post-Golgi

secretion

and exosome formation, a mech-

anism of unconventional secretion.

The

use of this model was based on the facts

that: (1) genes required for conventional,

post-Golgi secretion were implicated in

the formation of extracellular vesicles in

fungi;

and (2) exosomes and fungal vesi-

cles share many similarities.

18-20,23,24

Defects in the formation of multive-

sicular bodies (MVB) are expected to

directly affect the formation of exosomes.

Surprisingly, yeast mutants with defects

in MVB formation produced similar

amounts of extracellular vesicles in

comparison to WT cells.

The protein

composition of vesicles from WT and

mutant cells was essentially equivalent,

but approximately 50% of these vesicu-

lar proteins had their abundance modi-

ﬁed in mutant cells. Remarkably, most

of the proteins (75%) found in vesicular

fractions lacked signal peptides. These

puzzling results indicate that, although

MVB-related mutations apparently do

not affect vesicle release, MVB formation

is somehow related to the formation of

extracellular vesicles in yeast.

Post-Golgi secretory vesicles usually

fuse with the plasma membrane to release

Figure 1. Cryptococcus neoformans, a yeast pathogen, produce vesicle-like structures (arrows) that

apparently bud from the plasma membrane to be deposited at the cell wall, as evidenced from

transmission electron microscopy. Gold labeling represents reactivity of fungal glucosylceramide

with human antibodies. Scale bar, 0.1 µm. Asterisk denotes the cell wall. For experimental details,

see Rodrigues and colleagues.

Modied from Barreto-Bergter et al.

courtesy of Dr. Kildare

Miranda.

www.landesbioscience.com Communicative & Integrative Biology 535

17. van Niel G, Porto-Carreiro I, Simoes S, Raposo G.

Exosomes: a common pathway for a specialized func-

tion. J Biochem 2006; 140:13-21.

18. Aoki N, Jin-no S, Nakagawa Y, Asai N, Arakawa E,

Tamura N, et al. Identification and characterization

of microvesicles secreted by 3T3-L1 adipocytes:

redox- and hormone-dependent induction of milk

fat globule-epidermal growth factor 8-associated

microvesicles. Endocrinology 2007; 148:3850-62.

19. Gatti JL, Metayer S, Belghazi M, Dacheux F,

Dacheux JL. Identification, proteomic profiling and

origin of ram epididymal fluid exosome-like vesicles.

Biol Reprod 2005; 72:1452-65.

20. Mears R, Craven R A, Hanrahan S, Totty N, Upton

C, Young SL, et al. Proteomic analysis of melanoma-

derived exosomes by two-dimensional polyacryl-

amide gel electrophoresis and mass spectrometry.

Proteomics 2004; 4:4019-31.

21. Oliveira DL, Nakayasu ES, Joffe LS, Guimarães AJ,

Sobreira TJP, Nosanchuk JD, et al. Characterization

of yeast extracellular vesicles: evidence for the par-

ticipation of different pathways of cellular traffic in

vesicle biogenesis. PLoS ONE 2010; 5:11113.

22. Panepinto J, Komperda K, Frases S, Park YD,

Djordjevic JT, Casadevall A, et al. Sec6-dependent

sorting of fungal extracellular exosomes and lac-

case of Cryptococcus neoformans. Mol Microbiol 2009;

71:1165-76.

23. Valadi H, Ekstrom K, Bossios A, Sjostrand M,

Lee JJ, Lotvall JO. Exosome-mediated transfer of

mRNAs and microRNAs is a novel mechanism of

genetic exchange between cells. Nat Cell Biol 2007;

9:654-9.

24. Nicola AM, Frases S, Casadevall A. Lipophilic dye

staining of Cryptococcus neoformans extracellular vesi-

cles and capsule. Eukaryot Cell 2009; 8:1373-80.

25. Barreto-Bergter E, Pinto MR, Rodrigijes ML, Atta-

ur R. Structural and functional aspects of fun-

gal glycosphingolipids. Studies in Natural Products

Chemistry: Elsevier 2006; 1025-55.

26. Rodrigues ML, Travassos LR, Miranda KR, Franzen

AJ, Rozental S, de Souza W, et al. Human antibodies

against a purified glucosylceramide from Cryptococcus

neoformans inhibit cell budding and fungal growth.

Infect Immun 2000; 68:7049-60.

27. Duran JM, Anjard C, Stefan C, Loomis WF,

Malhotra V. Unconventional secretion of Acb1 is

mediated by autophagosomes. J Cell Biol 2010;

188:527-36.

28. Manjithaya R, Anjard C, Loomis WF, Subramani S.

Unconventional secretion of Pichia pastoris Acb1 is

dependent on GR ASP protein, peroxisomal functions

and autophagosome formation. J Cell Biol 2010;

188:537-46.

29. Abrahamsen H, Stenmark H. Protein secretion:

unconventional exit by exophagy. Curr Biol 2010;

20 : 415-8.

30. Kinseth MA, Anjard C, Fuller D, Guizzunti G,

Loomis WF, Malhotra V. The Golgi-associated protein

GRASP is required for unconventional protein secre-

tion during development. Cell 2007; 130:524-34.

31. De Jesus M, Nicola AM, Rodrigues ML, Janbon

G, Casadevall A. Capsular localization of the

Cryptococcus neoformans polysaccharide component

galactoxylomannan. Eukaryot Cell 2009; 8:96-103.

32. Oliveira DL, Nimrichter L, Miranda K, Frases S,

Faull KF, Casadevall A, et al. Cryptococcus neoformans

cryoultramicrotomy and vesicle fractionation reveals

an intimate association between membrane lipids

and glucuronoxylomannan. Fungal Genet Biol 2009;

46:956-63.

33. Eisenman HC, Frases S, Nicola AM, Rodrigues

ML, Casadevall A. Vesicle-associated melanization

in Cryptococcus neoformans. Microbiology 2009;

155 : 38 6 0 -7.

Jorge J. Jó Bastos Ferreira for his invaluable

suggestions on the role of vesicles in fun-

gal physiology. We are also grateful to the

Biomolecule Analysis Core Facility, Border

Biomedical Research Center, UTEP, funded

by NIH/NCRR grant 5G12RR008124-

16A1 and 5G12RR008124-16A1S1, for

continuous access to mass spectrometry

(LC-MS and GC-MS) instruments, which

have been fundamental for several of the

studies described here.

References

1. Jeremic A. Cell secretion: an update. J Cell Mol Med

2008; 12:1151-4.

2. Lee MC, Miller EA, Goldberg J, Orci L, Schekman

R. Bi-directional protein transport between the ER

and Golgi. Annu Rev Cell Dev Biol 2004; 20:87-

123.

3. Levi SK, Glick BS. GRASPing unconventional secre-

tion. Cell 2007; 130:407-9.

4. Nickel W, Rabouille C. Mechanisms of regulated

unconventional protein secretion. Nat Rev Mol Cell

Biol 2009; 10:148-55.

5. Herrmann JM, Malkus P, Schekman R. Out of the

ER—outfitters, escorts and guides. Trends Cell Biol

1999; 9:5-7.

6. Schekman RW. Regulation of membrane traffic in

the secretory pathway. Harvey Lect 1994; 90:41-57.

7. Rothblatt J, Schekman R. A hitchhiker’s guide to

analysis of the secretory pathway in yeast. Methods

Cell Biol 1989; 32:3-36.

8. Pugsley AP, Francetic O, Driessen AJ, de Lorenzo

V. Getting out: protein traffic in prokaryotes. Mol

Microbiol 2004; 52:3-11.

9. Nosanchuk JD, Nimrichter L, Casadevall A,

Rodrigues ML. A role for vesicular transport of mac-

romolecules across cell walls in fungal pathogenesis.

Commun Integr Biol 2008; 1:37-9.

10. Rodrigues ML, Nimrichter L, Oliveira DL,

Nosanchuk JD, Casadevall A. Vesicular trans-cell

wall transport in fungi: a mechanism for the deliv-

ery of virulence-associated macromolecules? Lipid

Insights 2008; 2008:27.

11. Casadevall A, Nosanchuk JD, Williamson P,

Rodrigues ML. Vesicular transport across the fungal

cell wall. Trends Microbiol 2009; 17:158-62.

12. Rodrigues ML, Nimrichter L, Oliveira DL, Frases

S, Miranda K, Zaragoza O, et al. Vesicular polysac-

charide export in Cryptococcus neoformans is a eukary-

otic solution to the problem of fungal trans-cell wall

transport. Eukaryot Cell 2007; 6:48-59.

13. Rodrigues ML, Nakayasu ES, Oliveira DL,

Nimrichter L, Nosanchuk JD, Almeida IC, et al.

Extracellular vesicles produced by Cryptococcus neo-

formans contain protein components associated with

virulence. Eukaryot Cell 2008; 7:58-67.

14. Albuquerque PC, Nakayasu ES, Rodrigues ML,

Frases S, Casadevall A, Zancope-Oliveira RM, et al.

Vesicular transport in Histoplasma capsulatum: an

effective mechanism for trans-cell wall transfer of

proteins and lipids in ascomycetes. Cell Microbiol

2008; 10:1695-710.

15. Oliveira DL, Freire-de-Lima CG, Nosanchuk

JD, Casadevall A, Rodrigues ML, Nimrichter L.

Extracellular vesicles from Cryptococcus neoformans

modulate macrophage functions. Infect Immun

2010; 78:1601-9.

16. Regente M, Corti-Monzon G, Maldonado AM,

Pinedo M, Jorrin J, de la Canal L. Vesicular fractions

of sunflower apoplastic fluids are associated with

potential exosome marker proteins. FEBS Lett 2009;

583:3363-6.

release has been associated to protein and

polysaccharide secretion,

12-14,22,32

surface

architecture,

virulence,

10,12,13

pigmenta-

tion

and modulation of macrophage

function.

Despite their apparent multiple

functions in yeast, the cellular components

controlling their biogenesis and release

remain elusive. We emphasize the sup-

position that the methods currently used

for preparation of extracellular fractions

containing vesicles may co-isolate vesicu-

lar compartments of different cellular ori-

gins, which limit the application of studies

based on the generation of punctual muta-

tions. Post-Golgi components required for

conventional secretion, proteins involved

in MVB formation, GRASP and even

autophagy-related events may be involved

in the formation of extracellular vesicles.

Although much progress has been made in

the last three years, the route to understand

how fungal extracellular vesicles are formed

still seems long and laborious.

Acknowledgements

D.L.O. is a Ph.D. student at Instituto de

Bioquímica Médica (Federal University

of Rio de Janeiro, Brazil). L.N. and

M.L.R. are supported by grants from the

Brazilian agencies Conselho Nacional de

Desenvolvimento Cientíﬁco e Tecnológico

(CNPq), Fundação de Amparo à Pesquisa

do Estado do Rio de Janeiro (FAPERJ)

and Coordenação de Aperfeiçoamento de

Pessoal de Nível Superior (CAPES). A.C.

is supported by NIH awards HL059842,

AI033774, AI052733, AI033142. A.J.G.

and L.N. were supported in part by an

Interhemispheric Research Training Grant

in Infectious Diseases, Fogarty Inter-

national Center (NIH D43-TW007129).

J.D.N. is supported in part by NIH

AI52733-06A1 and AI056070-01A2 and

a Hirschl/Weill-Caulier Career Scientist

Award. R.J.B.C. was supported by the

Training Program in Cellular and Molecular

Biology and Genetics, T32 GM007491.

I.C.A. was partially supported by the NIH/

NCRR grant 5G12RR008124-16A1 and

5G12RR008124-16A1S1. E.S.N was par-

tially supported by the George A. Krutilek

memorial graduate scholarship from

Graduate School, University of Texas at

El-Paso (UTEP). T.J.P.S. was supported by

Fundação de Amparo à Pesquisa do Estado

de São Paulo (FAPESP), Brazil. We thank

103

Anexo 4 – Supporting Information S1:

Protein

Description

Cellular distribution

metabolic process

YLR134W

Pdc5p, minor isoform of pyruvate decarboxylase

Nucleus, cytoplasm

YGR087C

Pdc6p, minor isoform of pyruvate decarboxylase

Cytoplasm

YKL152C

Gpm1p, tetrameric phosphoglycerate mutase

Cytosol, mitochondria

YML028W

Tsa1p, ubiquitous housekeeping thioredoxin peroxidase

Cytoplasm

YPR145W

Asn1p, asparagine synthetase

Cytoplasm

YLR303W

Met17p, methionine and cysteine synthase

Cytoplasm

YJL153C

Ino1p, inositol 1-phosphate synthase

Cytoplasm

YDR304C

Cpr5p, peptidyl-prolyl cis-trans isomerase (cyclophilin) of the

endoplasmic reticulum

Endoplasmic reticulum,

cytoplasm

YLR299W

Ecm38p, gamma-glutamyltranspeptidase

Cytoplasm, plasma

membrane

YDR483W

Kre2p, alpha1,2-mannosyltransferase of the Golgi involved in

protein mannosylation

Golgi apparatus, plasma

membrane, Golgi stack

YGR192C

Tdh3p, glyceraldehyde-3-phosphate dehydrogenase

Cytoplasm, mitochondria,

cell wall

YLR109W

Ahp1p, thiol-specific peroxiredoxin

Peroxisome, plasma

membrane, cytoplasm

YGR180C

Rnr4p, ribonucleotide-diphosphate reductase (RNR), small

subunit

Ribonucleoside-

diphosphate reductase,

nucleus, cytoplasm

YHR183W

Gnd1p, 6-phosphogluconate dehydrogenase

Mitochondria, cytoplasm

YHR179W

Oye2p, Widely conserved NADPH oxidoreductase containing

flavin mononucleotide (FMN), may be involved in sterol

metabolism

Nucleus, mitochondria,

cytoplasm

YGL234W

Ade5,7p, Bifunctional enzyme of the 'de novo' purine

nucleotide biosynthetic pathway

Cytoplasm

104

YOL011W

Plb3p, Phospholipase B (lysophospholipase); hydrolyzes

phosphatidylinositol and phosphatidylserine

Extracellular region,

plasma membrane

YLR245C

Cdd1p, Cytidine deaminase; catalyzes the modification of

cytidine to uridine

Nucleus, cytoplasm

YDL124W

Ydl124wp, NADPH-dependent alpha-keto amide reductase

Nucleus, cytoplasm

YML126C

Erg13p, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase

Endoplasmic reticulum,

mitochondria

YDR050C

Tpi1p, Triose phosphate isomerase, abundant glycolytic

enzyme.

Cytosol, mitochondria,

Cytoplasm

YLR044C

Pdc1p, Major of three pyruvate decarboxylase isozymes, key

enzyme in alcoholic fermentation.

Nucleus, cytoplasm

YGL202W

Aro8p, Aromatic aminotransferase I

Cytoplasm

YDR044W

Hem13p, Coproporphyrinogen III oxidase, catalyzes the sixth

step in the heme biosynthetic pathway

Mitochondrial inner

membrane

YEL047C

Yel047cp, Soluble fumarate reductase

Cytosol, ribosome,

mitochondria

YKL060C

Fba1p, Fructose 1,6-bisphosphate aldolase

Cytosol, mitochondria,

cytoplasm

YBR199W

Ktr4p, Putative mannosyltransferase involved in protein

glycosylation; member of the KRE2/MNT1

mannosyltransferase family

Golgi apparatus, integral to

membrane, membrane

YBR196C

Pgi1p, Glycolytic enzyme phosphoglucose isomerase

Cytosol, mitochondria

YCR012W

Pgk1p, 3-phosphoglycerate kinase; key enzyme in glycolysis

and gluconeogenesis

Cytosol, mitochondria,

cytoplasm

YJL052W

Tdh1p, Glyceraldehyde-3-phosphate dehydrogenase, isozyme

1, involved in glycolysis and gluconeogenesis

Lipid particle, Cell wall,

cytosol, mitochondria,

cytoplasm

YJR009C

Tdh2p, Glyceraldehyde-3-phosphate dehydrogenase, isozyme

2, involved in glycolysis and gluconeogenesis

Lipid particle, Cell wall,

cytosol, mitochondria,

cytoplasm

105

YAL038W

Cdc19p, Pyruvate kinase

Cytosol

YMR303C

Adh2p, Glucose-repressible alcohol dehydrogenase II

Cytoplasm

YGR254W

Eno1p, Enolase I, a phosphopyruvate hydratase

Mitochondria, vacuole,

cytoplasm

YOR133W

Eft1p, Elongation factor 2 (EF-2), catalyzes ribosomal

translocation during protein synthesis

Ribosome

YER091C

Met6p, Cobalamin-independent methionine synthase,

involved in amino acid biosynthesis

Cytoplasm

YCL043C

Pdi1p, Protein disulfide isomerase, multifunctional protein

resident in the endoplasmic reticulum lumen, essential for the

formation of disulfide bonds in secretory and cell-surface

proteins

Endoplasmic reticulum,

endoplasmic reticulum

lumen

YAL012W

Cys3p, Cystathionine gamma-lyase, catalyzes one of the two

reactions involved in the transsulfuration pathway that yields

cysteine from homocysteine

Cytoplasm

YDR533C

Hsp31p, Possible chaperone and cysteine protease

Soluble fraction

YDL045C

Fad1p, Flavin adenine dinucleotide (FAD) synthetase

Cytoplasm

YJR148W

Bat2p, Cytosolic branched-chain amino acid aminotransferase

Nucleus, cytoplasm

YHR057C

Cpr2p, Peptidyl-prolyl cis-trans isomerase (cyclophilin),

catalyzes the cis-trans isomerization of peptide bonds N-

terminal to proline residues; has a potential role in the

secretory pathway

Cellular component

unknown

YOR099W

Ktr1p, Alpha-1,2-mannosyltransferase involved in O- and N-

linked protein glycosylation

Golgi apparatus,

integral to membrane,

membrane, Golgi stack

YJR139C

Hom6p, Homoserine dehydrogenase (L-homoserine:NADP

oxidoreductase), dimeric enzyme that catalyzes the third step

in the common pathway for methionine and threonine

biosynthesis;

Nucleus, cytoplasm

YHR008C

Sod2p, Mitochondrial superoxide dismutase, protects cells

against oxygen toxicity

Mitochondrial matrix,

mitochondria

106

YER043C

Sah1p, S-adenosyl-L-homocysteine hydrolase

Cytoplasm

YNL117W

Mls1p, Malate synthase, enzyme of the glyoxylate cycle,

involved in utilization of non-fermentable carbon sources

Peroxisomal matrix,

glyoxysome, cytoplasm

YKL216W

Ura1p, Dihydroorotate dehydrogenase, catalyzes the fourth

enzymatic step in the de novo biosynthesis of pyrimidines

Extrinsic to membrane,

cytoplasm

YOR375C

Gdh1p, NADP(+)-dependent glutamate dehydrogenase

Nucleus, cytoplasm

YHR174W

Eno2p, Enolase II, a phosphopyruvate hydratase activity

Soluble fraction, vacuole,

mitochondria

YHR208W

Bat1p, Mitochondrial branched-chain amino acid

aminotransferase

Mitochondrial matrix,

mitochondria

YJL130C

Ura2p, Bifunctional carbamoylphosphate synthetase (CPSase)-

aspartate transcarbamylase (ATCase), catalyzes the first two

enzymatic steps in the de novo biosynthesis of pyrimidines

Mitochondria, cytoplasm,

integral to membrane

YAR071W

Pho11p, One of three repressible acid phosphatases, a

glycoprotein that is transported to the cell surface by the

secretory pathway

Extracellular region

YBR011C

Ipp1p, Cytoplasmic inorganic pyrophosphatase (PPase)

Cytosol

YJL026W

Rnr2p, Ribonucleotide-diphosphate reductase (RNR), small

subunit

Nucleus, cytoplasm

YGR256W

Gnd2p, 6-phosphogluconate dehydrogenase (decarboxylating),

catalyzes an NADPH regenerating reaction in the pentose

phosphate pathway

Cytosol

YOL086C

Adh1p, Alcohol dehydrogenase, fermentative isozyme

Cytosol

YKL157W

Ape2p, Zinc-dependent metallopeptidase yscII

Mitochondria, cell wall-

bounded periplasmic

space, cytoplasm

cell organization and biogenesis

YPL053C

Ktr6p, Probable mannosylphosphate transferase involved in

the synthesis of core oligosaccharides in protein glycosylation

pathway

Membrane fraction,

integral to membrane

107

YGR032W

Gsc2p, Catalytic subunit of 1,3-beta-glucan synthase, involved

in formation of the inner layer of the spore wall

Prospore membrane, 1,3-

beta-glucan synthase

complex, membrane

fraction, integral to

membrane, actin cap

YLR342W

Fks1p, Catalytic subunit of 1,3-beta-D-glucan synthase,

involved in cell wall synthesis and maintenance; localizes to

sites of cell wall remodeling

1,3-beta-glucan synthase

complex, integral to

membrane, actin cortical

patch, actin cap,

mitochondria

YLR355C

Ilv5p, Acetohydroxyacid reductoisomerase, mitochondrial

protein involved in amino acid biosynthesis, also required for

maintenance of mitochondrial DNA

Mitochondria,

mitochondrial nucleoid

YMR307W

Gas1p, Beta-1,3-glucanosyltransferase, required for cell wall

assembly; localizes to the cell surface via a

glycosylphosphatidylinositol (GPI) anchor

Cell wall, plasma

membrane, mitochondria

YGR282C

Bgl2p, Endo-beta-1,3-glucanase, major protein involved in cell

wall maintenance

Cell wall

YDR055W

Pst1p, Cell wall protein that contains a putative GPI-

attachment site; secreted by regenerating protoplasts; up-

regulated by cell wall damage

Cell wall, Plasma

membrane

YLR249W

Yef3p, Translational elongation factor 3, stimulates the binding

of aminoacyl-tRNA (AA-tRNA) to ribosomes

Ribosome

YDR261C

Exg2p, Exo-1,3-beta-glucanase, involved in cell wall beta-

glucan assembly; may be anchored to the plasma membrane

via a glycosylphosphatidylinositol (GPI) anchor

Cell wall, membrane

YBR078W

Ecm33p, GPI-anchored protein of unknown function, has a

possible role in apical bud growth

Membrane fraction, Cell

wall, mitochondria

YJR105W

Ado1p, Adenosine kinase, may be involved in recycling of

adenosine

Nucleus, cytoplasm

YLR300W

Exg1p, Major exo-1,3-beta-glucanase involved in cell wall beta-

glucan assembly

Cell wall

YLR121C

Yps3p, Aspartic protease, attached to the plasma membrane

Anchored to plasma

108

via a glycosylphosphatidylinositol (GPI) anchor

membrane

YMR186W

Hsc82p, Cytoplasmic chaperone of the Hsp90 family,

redundant in function and nearly identical with Hsp82p

Mitochondria, cytoplasm

YFL039C

Act1p, Actin, structural protein involved in cell polarization,

endocytosis, and other cytoskeletal functions

Cytoplasm

YLL026W

Hsp104p, Heat shock protein that cooperates to refold and

reactivate previously denatured, aggregated proteins;

responsive to stresses including: heat, ethanol, and sodium

arsenite

Nucleus, cytoplasm

YPL106C

Sse1p, ATPase that is a component of the heat shock protein

Hsp90 chaperone complex

Cytoplasm

Transporters

YPL036W

Pma2p, Plasma membrane H+-ATPase, isoform of Pma1p,

regulator of cytoplasmic pH and plasma membrane potential

Mitochondria, plasma

membrane, Integral to

membrane

YGL253W

Hxk2p, Hexokinase isoenzyme 2 that catalyzes

phosphorylation of glucose in the cytosol

Cytosol, mitochondria,

nucleus

YCR053W

Thr4p, Threonine synthase, conserved protein that catalyzes

formation of threonine

Nucleus, cytoplasm

YPR080W

Tef1p,Translational elongation factor EF-1 alpha; functions in

the binding reaction of aminoacyl-tRNA (AA-tRNA) to

ribosomes

Eukaryotic translation

elongation factor 1

complex, ribosome,

mitochondria

YJR048W

Cyc1p, Cytochrome c, isoform 1; electron carrier of the

mitochondrial intermembrane space

Mitochondrial

intermembrane space

Mitochondrial respiratory

chain

YGL008C

Pma1p, Plasma membrane H+-ATPase, major regulator of

cytoplasmic pH and plasma membrane potential

Lipid raft, mitochondria,

plasma membrane

YFR053C

Hxk1p, Hexokinase isoenzyme 1

Cytosol

YDL046W

Npc2p , Functional homolog of human NPC2/He1

Vacuole, cell cycle-

correlated morphology

109

YEL039C

Cyc7p, Cytochrome c isoform 2; electron carrier of the

mitochondrial intermembrane space

Mitochondrial respiratory

chain

carbohydrate metabolism

YGL028C

Scw11p , Cell wall protein with similarity to glucanases; may

play a role in conjugation during mating

Cell wall

YGR279C

Scw4p, Cell wall protein with similarity to glucanases

Cell wall

YNR067C

Dse4p, Daughter cell-specific secreted protein with similarity

to glucanases, degrades cell wall from the daughter side

causing daughter to separate from mother

Extracellular region, Cell

wall

YMR105C

Pgm2p, Phosphoglucomutase, catalyzes a key step in hexose

metabolism

Cytosol, cytoplasm

YLR354C

Tal1p, Transaldolase, enzyme in the non-oxidative pentose

phosphate pathway

Cytoplasm

YMR305C

Scw10p, Cell wall protein with similarity to glucanases; may

play a role in conjugation during mating

Endoplasmic reticulum,

Cell wall, cytoplasm

response to stress

YMR169C

Ald3p, Cytoplasmic aldehyde dehydrogenase, involved in beta-

alanine synthesis

Cytoplasm

YGR234W

Yhb1p, Nitric oxide oxidoreductase, flavohemoglobin involved

in nitric oxide detoxification; plays a role in the oxidative and

nitrosative stress responses

Mitochondrial matrix,

cytoplasm, cytosol

YDL229W

Ssb1p, Cytoplasmic ATPase that is a ribosome-associated

molecular chaperone

Soluble fraction, polysome

YNL160W

Ygp1p , Cell wall-related secretory glycoprotein; induced by

nutrient deprivation-associated growth arrest and upon entry

into stationary phase

Cell wall

YNL209W

Ssb2p, Cytoplasmic ATPase that is a ribosome-associated

molecular chaperone

Polysome, cytoplasm

YPL240C

Hsp82p, Hsp90 chaperone required for pheromone signaling

Cytoplasm

Protein biosynthesis

110

YLR075W

Rpl10p , Protein component of the large (60S) ribosomal

subunit

Cytosolic large ribosomal

subunit

YBR118W

Tef2p,Translational elongation factor EF-1 alpha; functions in

the binding reaction of aminoacyl-tRNA (AA-tRNA) to

ribosomes

Eukaryotic translation

elongation factor 1

complex, ribosome,

cytoplasm

YDR012W

Rpl4bp, Protein component of the large (60S) ribosomal

subunit

Cytosolic large ribosomal

subunit, cytoplasm

YDR385W

Eft2p, Elongation factor 2 (EF-2); catalyzes ribosomal

translocation during protein synthesis

Ribosome

YBR031W

Rpl4ap, N-terminally acetylated protein component of the

large (60S) ribosomal subunit

Cytosolic large ribosomal

subunit

YJR123W

Rps5p, Protein component of the small (40S) ribosomal

subunit

Cytosolic small ribosomal

subunit

protein degradation

YCL057W

Prd1, Zinc metalloendopeptidase, involved in degradation of

mitochondrial proteins and of presequence peptides cleaved

from imported proteins

Mitochondrial

intermembrane space,

Cytoplasm

YMR297W

Prc1p, Vacuolar carboxypeptidase Y (proteinase C), broad-

specificity C-terminal exopeptidase involved in non-specific

protein degradation in the vacuole; member of the serine

carboxypeptidase family; protein coding

Lumen of vacuole with cell

cycle-correlated

morphology, endoplasmic

reticulum, cytoplasm

YPL235W

Rvb2p, Essential protein involved in transcription regulation;

component of chromatin remodeling complexes

Nucleus, chromatin

remodeling complex,

SWR1 complex, INO80

complex

YFR044C

Dug1p, Probable di- and tri-peptidase; forms a complex to

degrade glutathione (GSH)

Ribosome, mitochondria,

cytoplasm

YGL203C

Kex1p, Protease involved in the processing of killer toxin and

alpha factor precursor; cleaves Lys and Arg residues from the

C-terminus of peptides and proteins; protein coding

Trans-Golgi network,

integral to membrane

protein transport

111

YJL034W

Kar2p , ATPase involved in protein import into the ER, also acts

as a chaperone to mediate protein folding in the ER and may

play a role in ER export of soluble proteins

Endoplasmic reticulum

lumen, endoplasmic

reticulum

YLL024C

Ssa2p , ATP binding protein involved in protein folding and

vacuolar import of proteins

Membrane of vacuole with

cell cycle-correlated

morphology, chaperonin-

containing T-complex, Cell

wall, mitochondria,

cytoplasm

YBL075C

Ssa3p, ATPase involved in protein folding and the response to

stress; plays a role in SRP-dependent cotranslational protein-

membrane targeting and translocation; member of the heat

shock protein 70 (HSP70) family

Cytosol

YAL005C

Ssa1p, ATPase involved in protein folding and nuclear

localization signal (NLS)-directed nuclear transport; member of

heat shock protein 70 (HSP70) family

Membrane of vacuole with

cell cycle-correlated

morphology, chaperonin-

containing T-complex, Cell

wall, nucleus, cytoplasm

YER103W

Ssa4p, Heat shock protein that is highly induced upon stress;

plays a role in SRP-dependent cotranslational protein-

membrane targeting and translocation

Nucleus, cytoplasm

Sporulation

YPL154C

Pep4p, Vacuolar aspartyl protease (proteinase A), required for

the posttranslational precursor maturation of vacuolar

proteinases; important for protein turnover after oxidative

damage

Lumen of vacuole with cell

cycle-correlated

morphology, mitochondria

YDR155C

Cpr1p, Cytoplasmic peptidyl-prolyl cis-trans isomerase

(cyclophilin), catalyzes the cis-trans isomerization of peptide

bonds N-terminal to proline residues

Histone deacetylase

complex, mitochondria,

nucleus

YER177W

Bmh1p, 14-3-3 protein, major isoform; controls proteome at

post-transcriptional level, binds proteins and DNA, involved in

regulation of many processes including exocytosis, vesicle

transport, Ras/MAPK signaling, and rapamycin-sensitive

signaling

Nucleus

YDR099W

Bmh2p, 14-3-3 protein, minor isoform; controls proteome at

Nucleus

112

post-transcriptional level, binds proteins and DNA, involved in

regulation of many processes including exocytosis, vesicle

transport, Ras/MAPK signaling, and rapamycin-sensitive

signaling

Unknown

YJL171C

Yjl171cp, GPI-anchored cell wall protein of unknown function;

induced in response to cell wall damaging agents and by

mutations in genes involved in cell wall biogenesis

Cell wall, mitochondria

YNL134C

Ynl134cp, Putative protein of unknown function with similarity

to dehydrogenases from other model organisms

Nucleus, cytoplasm

YLR178C

Tfs1p, Carboxypeptidase Y inhibitor,

phosphatidylethanolamine-binding protein involved in protein

kinase A signaling pathway

Membrane of vacuole with

cell cycle-correlated

morphology, soluble

fraction, lumen of vacuole

with cell cycle-correlated

morphology, cytoplasm

YLR179C

Ylr179cp, Protein of unknown function, transcription is

activated by paralogous proteins Yrm1p and Yrr1p along with

proteins involved in multidrug resistance

Nucleus, cytoplasm

YHR138C

Yhr138cp, Putative protein of unknown function; has similarity

to Pbi2p; double null mutant lacking Pbi2p and Yhr138p

exhibits highly fragmented vacuoles

Cellular component

unknown

YNL208W

Ynl208wp, Protein of unknown function; may interact with

ribosomes, based on co-purification experiments; authentic,

non-tagged protein is detected in purified mitochondria

Ribosome, mitochondria

YLR414C

Ylr414cp, Putative protein of unknown function;

transcriptional induced in response to cell wall damage;

YLR414C is not an essential gene

Cellular bud, cytoplasm

YDR262W

Ydr262wp, Putative protein of unknown function; induced in

response to the DNA-damaging agent MMS; gene expression

increases in response to Zymoliase treatment

Vacuole, cell cycle-

correlated morphology

YOL030W

Gas5p, 1,3-beta-glucanosyltransferase, has similarity to Gas1p;

localizes to the cell wall

Membrane fraction,

membrane, Cell wall

113

YMR215W

Gas3p, Putative 1,3-beta-glucanosyltransferase, has similarity

to Gas1p; localizes to the cell wall

Membrane fraction,

membrane, Cell wall

YPL225W

Ypl225wp, Protein of unknown function that may interact with

ribosomes

Ribosome, cytoplasm

YHR215W

Pho12p , One of three repressible acid phosphatases, a

glycoprotein that is transported to the cell surface by the

secretory pathway; nearly identical to Pho11p

Vacuole, cell cycle-

correlated morphology

* Proteins with less than 10 spectra after MS/MS analysis were not listed in this table.

118

V- Discussão:

Nesta tese e em estudos desenvolvidos por nosso grupo e outros autores (Rodrigues, et al.

2008; Panepinto, et al., 2009; Hu & Kronstad, et al. 2009) foi demonstrado que espécies de

fungos produzem vesículas extracelulares contendo bicamadas lipídicas morfologicamente

similares à exossomos de células mamíferas, com dimensões variando entre 60 e 300 nm. Este

novo mecanismo de secreção está implicado tanto em processos fisiológicos (como o

crescimento capsular) quanto na interação com célula hospedeira em C. neoformans,

comprovando que estas vesículas são biologicamente ativas. A caracterização de vesículas de

diversos mutantes em vias de tráfego sugere que a biogênese vesicular envolve componentes de

diferentes vias, tanto clássicas quanto não-convencionais, o que retrata a complexidade deste

processo secretório.

Fez-se necessário legitimar a existência das vesículas secretadas como um fenômeno

biológico, o que foi comprovado de três maneiras: (i) foi visto que a produção de vesículas é

dependente de viabilidade celular; (ii) experimentos de pulso e caça revelaram que componentes

vesiculares advêm do metabolismo celular e (iii) vesículas não incorporam componentes

exógenos presentes no meio de cultura e portanto não são formadas randomicamente pela

associação aleatória de lipídeos (Anexo 1). Posteriormente foi demonstrado que diversas

espécies de fungos são capazes de secretar vesículas, consolidando a existência de um

mecanismo de secreção vesicular comum aos fungos (Albuquerque, et al. 2008).

É sabido que o mutante de C. neoformans incapaz de expressar o produto do gene SAV1,

homólogo ao SEC4 de S. cerevisiae, apresenta acúmulo de vesículas citoplasmáticas contendo

119

GXM (Yoneda & Doering, 2006). Tais vesículas advêm do Aparato de Golgi e seguindo a via

secretória convencional, deveriam fundir-se à membrana plasmática liberando a GXM no

periplasma (Ponnambalam & Baldwin, 2003). Este modelo, entretanto, confronta-se com o fato

de vesículas intactas contendo GXM serem liberadas no ambiente extracelular (Anexo 1, Figura

4A). Mais do que isso, o modelo de secreção de GXM proposto por Yoneda e Doering não explica

de que forma o polissacarídeo de alto peso molecular atravessa a densa estrutura da parede

celular (Yoneda & Doering, 2006). É possível que após a síntese no Aparato de Golgi, vesículas

brotem da TGN carreando GXM e direcionem-se a uma via não- convencional de secreção ao

invés de se fundirem a membrana plasmática. De fato, compartimentos semelhantes à MVBs de

células mamíferas foram encontrados no citoplasma do C. neoformans (Rodrigues, et al. 2008

Takeo, et al. 1973). Além disso, experimentos de crioultramicrotomia seguidos de imuno-

marcação revelaram que GXM é transportada por diferentes compartimentos potencialmente

incluindo vacúolos, estruturas reticulares e vesículas citoplasmáticas (Anexo 2, Figura 3), o que

sugere que a GXM deve passar por vias de tráfego mais complexas do que a exocitose

convencional.

Os mecanismos que medeiam a passagem de vesículas pela parede celular de fungos são

ainda pouco conhecidos. Estudos de microscopia de força atômica revelaram a existência de

poros na parede celular da levedura S. cerevisiae com diâmetro variando entre 200-400 nm (de

Souza Pereira & Geibel, 1999), o que comportaria a passagem das vesículas. Ainda assim

permanece em aberto a questão de como as vesículas seriam transportadas através destes

poros. Especula-se que proteínas motoras possam estar envolvidas em tal evento. É sabido que

miosina e actina estão envolvidas em vários processos associados à parede celular, como

120

formação de septo, crescimento na hifa, germinação e esporulação (Oberholzer, et al. 2002; Bi,

2001; Esnault, et al. 1999). Entretanto, ainda não existem evidências da participação direta de

proteínas envolvidas em trânsito de moléculas e organelas citoplasmáticas agindo na matriz da

parede celular.

Análises feitas por microscopia eletrônica de transmissão revelaram a existência de grupos de

vesículas com elétron densidades distintas (Rodrigues, et al. 2008). Esta observação nos levou a

questionar se tal diferença morfológica seria reflexo de vias distintas de biogênese. O

fracionamento vesicular em gradiente de densidade evidenciou uma população de vesículas

enriquecida em GXM e outra população de vesículas contendo níveis mínimos do polissacarídeo

(Anexo 2, Figura 5). A existência de mais de uma população de vesículas que diferem em termos

de conteúdo, densidade e morfologia reforça, portanto, a hipótese de que mais de uma via de

biogênese deva estar implicada na produção desses compartimentos membranosos. Análises

genéticas necessárias para a identificação destas vias no C. neoformans estão em andamento,

sendo parte de estudos futuros dentro do nosso laboratório.

A caracterização proteômica de vesículas de C. neoformans revelou uma complexa

composição protéica incluindo chaperonas, proteínas de membrana, proteínas citoplasmáticas,

mitocondriais e até nucleares (Rodrigues, et al. 2008). Tal complexidade reflete uma

característica peculiar de fungos: proteínas localizam-se em mais de um compartimento celular

onde devem exercer funções distintas (Nimrichter, et al. 2005). Esse fenômeno, denominado

‘moonlighting’ pelo grupo de Jeffery (Jeffery, 1999), tem vários precedentes na área de Biologia

Celular de fungos. Em Histoplasma capsulatum, por exemplo, histonas são encontradas na

parede celular (Nosanchuk, et al. 2003). Em Paracoccidioides brasiliensis, a enzima glicolítica

121

gliceraldeído-3-fosfato desidrogenase também se encontra na parede celular, onde participa da

patogênese mediando adesão de leveduras a matriz extracelular (Barbosa, et al. 2006). A

proteína mitocondrial Mdj1 é também encontrada na parede celular em P. brasiliensis (Batista,

et al. 2006). É possível, portanto, que proteínas vesiculares de C. neoformans apresentem

funções no espaço extracelular, sobretudo em relação a patogênese, virulência e imunogênese.

De acordo com essa hipótese está o fato de que extratos de proteínas vesiculares reagem com

soro de pacientes infectados por C. neoformans (Rodrigues, et al. 2008), confirmando que as

mesmas são secretadas durante a infecção, funcionando como imunógenos ativadores da

resposta humoral.

Mecanismos de virulência e processos secretórios estão intimamente relacionados em

diversos modelos microbianos (Hauser, et al. 1998; Ghannoum, 1998; Hoegl, et al, 1996; Hube, et

al. 1996). Na presente tese foi demonstrado que vesículas de C. neoformans podem ser

internalizadas por macrófagos induzindo sua ativação, evento que leva a aumentos do índice de

fagocitose e de atividade microbicida (Anexo 3). Sabe-se que além de GXM, vesículas de C.

neoformans carreiam diversos outros moduladores de virulência como lacase, superóxido

dismutase, glucosilceramida e urease (Rodrigues, et al. 2008). No entanto estas estruturas não se

mostraram citotóxicas em modelo de linhagem de macrófagos (Anexo3, Figura 1C). O

empacotamento de diversas moléculas relacionadas à virulência dentro de uma mesma estrutura

poderia representar um mecanismo vantajoso de concentração de enzimas e moléculas

antioxidantes que agiriam na defesa da célula fúngica. Entretanto este coquetel de moléculas

fúngicas induz eficiente ativação celular e produção de NO e citocinas (Anexo 3). É possível que a

concentração de componentes do patógeno permita assimilação mais eficiente por parte do

122

fagócito do que se estas moléculas estivessem solúveis no meio, levando a uma resposta mais

eficiente na célula hospedeira. Preparações de TEM de pulmões de camundongos infectados

sugerem fortemente que vesículas são produzidas in vivo durante a infecção (Anexo 1, Figura 1).

É importante ressaltar que incubação de células com vesículas isoladas do sobrenadante de

cultivo não necessariamente mimetiza a produção de vesículas que ocorreria naturalmente

durante a infecção em termos de concentração vesicular, tempo de exposição e possivelmente

de composição.

É notório que a presença de GXM nas vesículas influencia diretamente o perfil de citocinas

secretadas e o resultado da interação entre C. neoformans e fagócitos. Macrófagos incubados

com vesículas isoladas do mutante acapsular produzem significativamente mais TNF-α do que os

incubados com vesículas de células capsuladas e ao mesmo tempo produzem significativamente

menos IL-10 (Anexo 3, Figura 3), o que corrobora com o conhecido papel antiinflamatório e

imunossupressor da GXM (Monari, et al. 2009; Lupo, et al. 2008; Monari, et al. 2006; Villena, et

al. 2008). Mais do que isso, células incubadas com vesículas do mutante acapsular apresentaram

uma maior capacidade fagocítica e um menor tempo de incubação necessário para visualização

da atividade microbicida quando comparadas com células ativadas com vesículas contendo GXM

(Anexo 3, Figuras 4 e 5). Tais resultados sugerem que vesículas isoladas do sobrenadante de

cultura de C. neoformans apresentam grande potencial para modular a resposta imune e o curso

da infecção, uma vez que células fagocíticas exercem papel crucial no controle da criptococose

(Miller & Mitchel, 1991). Estudos sobre exossomos derivados de células dendríticas revelaram

que estas estruturas são promissoras vacinas no tratamento antitumoral (Wolfers, et al. 2001).

Neste sentido, é razoável considerar o desenvolvimento de uma possível vacina a partir de

123

vesículas produzidas pelo C. neoformans como estratégia terapêutica, considerando o fato de

vesículas conterem várias proteínas imunogênicas (Rodrigues, et al. 2008).

Embora a secreção vesicular tenha sido primeiramente caracterizada em C. neoformans,

vários fatores tornam S. cerevisiae um modelo mais promissor para o estudo das vias de

biogênese vesicular em fungos. Sendo essa a primeira levedura a ter seu genoma seqüenciado,

além de ser essa a espécie eucariótica como maior volume de informações disponíveis com

relação a eventos da biologia celular em geral, várias vantagens técnicas se configuram no

modelo do S. cerevisiae. Encontra-se disponível, por exemplo, uma vasta coleção de mutantes

com defeitos em vias de tráfego celular com fenótipos já caracterizados (Schekman, et al. 1983;

Winzeler, et al. 1999; Giaever, et al. 2002). Em conseqüência disso optamos por utilizar S.

cerevisiae com o intuito de definir genes cujos produtos poderiam estar relacionados com

produção e/ou liberação de vesículas extracelulares. Utilizando os protocolos estabelecidos por

nosso grupo para C. neoformans, vesículas extracelulares foram isoladas do sobrenadante de

cultivo de S. cerevisiae, conforme visualizado por TEM (Anexo 4, Figura 1). Dois mutantes

principais foram utilizados como protótipos neste estudo: o mutante sec4-2 ts, o qual é afetado

na via secretora clássica e o mutante snf7



, o qual apresenta defeitos na formação de

exossomos. O gene SEC4 foi selecionado por ser homólogo do gene SAV1 de C. neoformans, cujo

silenciamento leva ao acúmulo de vesículas citoplasmáticas contendo GXM (Yoneda & Doering,

2006). O gene SNF7 foi escolhido com base no fato de que seu produto, a Snf7p, atua

diretamente na formação de vesículas internas durante a maturação do endossoma em MVB,

afetando portanto a formação de exossomos (Tu, et al. 1993). Outros mutantes usados incluíram

o gene BOS1, envolvido no transporte a partir do RE, SEC1, relacionado à fusão de vesículas

124

oriundas do Aparato de Golgi com a membrana plasmática, e VPS23, que codifica um dos

componentes do complexo ESCRT-I, responsável pelo endereçamento de proteínas ao

compartimento MVB (Schekman, et al. 1983; Babst, et al. 2000; Wuestehube, et al. 1996). Um

mutante incapaz de expressar o produto do gene GRH1 também foi analisado, uma vez que seu

envolvimento em um mecanismo de secreção não- convencional foi recentemente descrito em S.

cerevisiae (Duran, et al. 2010).

A secreção vesicular não foi completamente abolida em nenhum dos mutantes analisados, o

que pode indicar que múltiplas vias estejam implicadas neste processo. A secreção vesicular pelo

mutante sec 4-2 ts, entretanto, foi expressivamente diminuída (Anexo 4, Figura 6), o que indica a

participação de um componente da via de secreção clássica na geração de vesículas. Contudo, o

mecanismo pelo qual vesículas extracelulares surgem a partir de vesículas citoplasmáticas

oriundas do Aparato de Golgi permanece desconhecido. É possível que eventos posteriores

viabilizem topologicamente a formação de tais vesículas utilizando maquinarias de secreção não-

convencional. De fato, foi demonstrado neste estudo que células mutantes deficientes na

expressão do produto do gene GRH1, as quais são deficientes no funcionamento de uma via de

secreção não-convencional dependente de mecanismos de autofagia (Duran, et al. 2010),

também apresentam diminuição acentuada da secreção de vesículas (Anexo 4, Figura 7). A

influência de mecanismos autofágicos na secreção vesicular será avaliada futuramente pelo

nosso grupo através de estudos de secreção em mutantes ATG, já em processo de preparação.

A caracterização proteômica das vesículas de S. cerevisiae revelou uma composição bastante

complexa, incluindo ao menos 127 proteínas (Anexo 4, Figura 2 e Tabela suplementar S1). Esse

perfil composicional foi consistentemente similar em todas as cepas analisadas (3 diferentes

125

cepas selvagens e 5 diferentes mutantes) (Anexo 4, Figura 4). Muitas destas proteínas são

citoplasmáticas e não se correlacionam com processos secretórios. A presença dessas proteínas,

entretanto, (por exemplo: piruvato quinase e glucose-6- fosfato isomerase) é freqüentemente

descrita em exossomos de mamíferos (Schorey & Bhatnagar, 2008). Especula-se que essa

observação estaria relacionada ao fato de que as invaginações na membrana do compartimento

endocítico levam à incorporação aleatória de porções do citosol (Schorey & Bhatnagar, 2008). A

complexidade da composição proteômica das vesículas de S. cerevisiae está aparentemente

relacionada a interações biológicas múltiplas. Em nosso estudo, estimamos que ocorram 219

diferentes interações dentre as 127 proteínas descritas (Anexo 4, Figura 2B). Diversas enzimas

que hidrolisam componentes estruturais da parede celular também foram encontradas nas

vesículas, sugerindo que o processo de passagem de vesículas através da parede possa envolver

remodelamento dessa estrutura. A análise proteômica revelou ainda um enriquecimento

importante de proteínas GPI-ancoradas bem como de proteínas sintetizadas no RE em frações

vesiculares. É sabido que proteínas GPI ancoradas são enriquecidas em microdomínios lipídicos

de membrana, o que poderia sugerir pistas da origem intracelular dessas membranas. Sabe-se

que exossomos de origem endossomal, bem como sítios de saída do RE, são enriquecidos em tais

microdomínios (Subra, et al. 2007; Mayor & Riezman, 2004).

Mutações relacionadas à formação de MVB não alteraram a quantidade de vesículas

produzidas por S. cerevisiae, além de não afetar seu perfil protéico qualitativo. Análises semi-

quantitativas do proteoma das vesículas, entretanto, mostraram alterações drásticas na

abundância relativa de proteínas (Anexo 4, Figura 5), sugerindo que defeitos na via de formação

de MVBs afetam a composição de vesículas extracelulares. A via de secreção não-convencional

126

dependente da proteína Grh1 também parece convergir para via de secreção de exossomos

(Duran, et al. 2010). Os dados semi-quantitativos de proteínas juntamente com análises

quantitativas de lipídeos sugerem que componentes de múltiplas vias devem estar relacionados

à produção de vesículas extracelulares. A secreção de vesículas parece ser um processo

multifatorial complexo que possivelmente envolve vias redundantes.

Esta tese propõe a existência de um novo veículo de transporte de macromoléculas para o

ambiente extracelular em fungos. Tal achado gera diversos questionamentos sobre o impacto

deste processo na fisiologia celular e na patôgenese de fungos. Outros aspectos funcionais das

vesículas, como por exemplo em processos de comunicação célula-célula ou transferência lateral

de material genético, devem ser investigados. Sobretudo, os mecanismos subjacentes a secreção

vesicular e transporte através da parede celular ainda precisam ser esclarecidos. Em suma, o

presente trabalho abre potenciais novas áreas de investigação sobre estrutura da parede celular

de fungos, mecanismos virulência e rotas não convencionais de secreção de proteínas.

127

VI- Referências Bibliográficas:

Alam, S.L., and Sundquist, W.I. (2007). Structural biology: ESCRT service. Nature 447, 921-922.

Albuquerque, P.C., Nakayasu, E.S., Rodrigues, M.L., Frases, S., Casadevall, A., Zancope-Oliveira,

R.M., Almeida, I.C., and Nosanchuk, J.D. (2008). Vesicular transport in Histoplasma capsulatum:

an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell

Microbiol 10, 1695-1710.

Altfeld, M., Addo, M.M., Kreuzer, K.A., Rockstroh, J.K., Dumoulin, F.L., Schliefer, K., Leifeld, L.,

Sauerbruch, T., and Spengler, U. (2000). T(H)1 to T(H)2 shift of cytokines in peripheral blood of

HIV-infected patients is detectable by reverse transcriptase polymerase chain reaction but not by

enzyme-linked immunosorbent assay under nonstimulated conditions. J Acquir Immune Defic

Syndr 23, 287-294.

Alvarez, M., and Casadevall, A. (2006). Phagosome extrusion and host-cell survival after

Cryptococcus neoformans phagocytosis by macrophages. Curr Biol 16, 2161-2165.

Andrei, C., Dazzi, C., Lotti, L., Torrisi, M.R., Chimini, G., and Rubartelli, A. (1999). The secretory

route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related

vesicles. Mol Biol Cell 10, 1463-1475.

Andrei, C., Margiocco, P., Poggi, A., Lotti, L.V., Torrisi, M.R., and Rubartelli, A. (2004).

Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for

inflammatory processes. Proc Natl Acad Sci U S A 101, 9745-9750.

Anjard, C., and Loomis, W.F. (2005). Peptide signaling during terminal differentiation of

Dictyostelium. Proc Natl Acad Sci U S A 102, 7607-7611.

Arora, S., Hernandez, Y., Erb-Downward, J.R., McDonald, R.A., Toews, G.B., and Huffnagle, G.B.

(2005). Role of IFN-gamma in regulating T2 immunity and the development of alternatively

activated macrophages during allergic bronchopulmonary mycosis. J Immunol 174, 6346-6356.

128

Babst, M., Odorizzi, G., Estepa, E.J., and Emr, S.D. (2000). Mammalian tumor susceptibility gene

101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking.

Traffic 1, 248-258.

Barbosa, F.M., Fonseca, F.L., Figueiredo, R.T., Bozza, M.T., Casadevall, A., Nimrichter, L., and

Rodrigues, M.L. (2007). Binding of glucuronoxylomannan to the CD14 receptor in human A549

alveolar cells induces interleukin-8 production. Clin Vaccine Immunol 14, 94-98.

Barbosa, M.S., Bao, S.N., Andreotti, P.F., de Faria, F.P., Felipe, M.S., dos Santos Feitosa, L.,

Mendes-Giannini, M.J., and Soares, C.M. (2006). Glyceraldehyde-3-phosphate dehydrogenase of

Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular

matrix proteins and interaction with cells. Infect Immun 74, 382-389.

Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F.,

Ravazzola, M., Amherdt, M., and Schekman, R. (1994). COPII: a membrane coat formed by Sec

proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895-907.

Barr, F.A., Puype, M., Vandekerckhove, J., and Warren, G. (1997). GRASP65, a protein involved in

the stacking of Golgi cisternae. Cell 91, 253-262.

Batista, W.L., Matsuo, A.L., Ganiko, L., Barros, T.F., Veiga, T.R., Freymuller, E., and Puccia, R.

(2006). The PbMDJ1 gene belongs to a conserved MDJ1/LON locus in thermodimorphic

pathogenic fungi and encodes a heat shock protein that localizes to both the mitochondria and

cell wall of Paracoccidioides brasiliensis. Eukaryot Cell 5, 379-390.

Bauman, S.K., Nichols, K.L., and Murphy, J.W. (2000). Dendritic cells in the induction of protective

and nonprotective anticryptococcal cell-mediated immune responses. J Immunol 165, 158-167.

Beatty, W.L., and Russell, D.G. (2000). Identification of mycobacterial surface proteins released

into subcellular compartments of infected macrophages. Infect Immun 68, 6997-7002.

Beenhouwer, D.O., Shapiro, S., Feldmesser, M., Casadevall, A., and Scharff, M.D. (2001). Both Th1

and Th2 cytokines affect the ability of monoclonal antibodies to protect mice against

Cryptococcus neoformans. Infect Immun 69, 6445-6455.

129

Bendtsen, J.D., Nielsen, H., von Heijne, G., and Brunak, S. (2004). Improved prediction of signal

peptides: SignalP 3.0. J Mol Biol 340, 783-795.

Bhatnagar, S., and Schorey, J.S. (2007). Exosomes released from infected macrophages contain

Mycobacterium avium glycopeptidolipids and are proinflammatory. J Biol Chem 282, 25779-

25789.

Bhatnagar, S., Shinagawa, K., Castellino, F.J., and Schorey, J.S. (2007). Exosomes released from

macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro

and in vivo. Blood 110, 3234-3244.

Bi, E. (2001). Cytokinesis in budding yeast: the relationship between actomyosin ring function

and septum formation. Cell Struct Funct 26, 529-537.

Bingle, L.E., Bailey, C.M., and Pallen, M.J. (2008). Type VI secretion: a beginner's guide. Curr Opin

Microbiol 11, 3-8.

Blackstock, R., Buchanan, K.L., Adesina, A.M., and Murphy, J.W. (1999). Differential regulation of

immune responses by highly and weakly virulent Cryptococcus neoformans isolates. Infect

Immun 67, 3601-3609.

Blackstock, R., and Murphy, J.W. (1997). Secretion of the C3 component of complement by

peritoneal cells cultured with encapsulated Cryptococcus neoformans. Infect Immun 65, 4114-

4121.

Blackstock, R., and Murphy, J.W. (2004). Role of interleukin-4 in resistance to Cryptococcus

neoformans infection. Am J Respir Cell Mol Biol 30, 109-117.

Bock, J.B., Matern, H.T., Peden, A.A., and Scheller, R.H. (2001). A genomic perspective on

membrane compartment organization. Nature 409, 839-841.

Bonifacino, J.S., and Glick, B.S. (2004). The mechanisms of vesicle budding and fusion. Cell 116,

153-166.

130

Bonifacino, J.S., and Lippincott-Schwartz, J. (2003). Coat proteins: shaping membrane transport.

Nat Rev Mol Cell Biol 4, 409-414.

Bose, I., Reese, A.J., Ory, J.J., Janbon, G., and Doering, T.L. (2003). A yeast under cover: the

capsule of Cryptococcus neoformans. Eukaryot Cell 2, 655-663.

Bowers, K., and Stevens, T.H. (2005). Protein transport from the late Golgi to the vacuole in the

yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1744, 438-454.

Cantin, R., Diou, J., Belanger, D., Tremblay, A.M., and Gilbert, C. (2008). Discrimination between

exosomes and HIV-1: purification of both vesicles from cell-free supernatants. J Immunol

Methods 338, 21-30.

Casadevall, A.,Perfect, J.R. (1998). Cryptococcus neoformans, American Society for Microbiology,

Washington.

Chaffin, W.L., Lopez-Ribot, J.L., Casanova, M., Gozalbo, D., and Martinez, J.P. (1998). Cell wall and

secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol

Rev 62, 130-180.

Chaka, W., Scharringa, J., Verheul, A.F., Verhoef, J., Van Strijp, A.G., and Hoepelman, I.M. (1995).

Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human

peripheral blood mononuclear cells by flow cytometry. Clin Diagn Lab Immunol 2, 753-759.

Chang, Y.C., and Kwon-Chung, K.J. (1994). Complementation of a capsule-deficient mutation of

Cryptococcus neoformans restores its virulence. Mol Cell Biol 14, 4912-4919.

Chang, Y.C., and Kwon-Chung, K.J. (1998). Isolation of the third capsule-associated gene, CAP60,

required for virulence in Cryptococcus neoformans. Infect Immun 66, 2230-2236.

Chang, Y.C., and Kwon-Chung, K.J. (1999). Isolation, characterization, and localization of a

capsule-associated gene, CAP10, of Cryptococcus neoformans. J Bacteriol 181, 5636-5643.

131

Chang, Y.C., Penoyer, L.A., and Kwon-Chung, K.J. (1996). The second capsule gene of

cryptococcus neoformans, CAP64, is essential for virulence. Infect Immun 64, 1977-1983.

Charlier, C., Nielsen, K., Daou, S., Brigitte, M., Chretien, F., and Dromer, F. (2009). Evidence of a

role for monocytes in dissemination and brain invasion by Cryptococcus neoformans. Infect

Immun 77, 120-127.

Chayakulkeeree, M., and Perfect, J.R. (2006). Cryptococcosis. Infect Dis Clin North Am 20, 507-

544, v-vi.

Chen, L.C., Goldman, D.L., Doering, T.L., Pirofski, L., and Casadevall, A. (1999). Antibody response

to Cryptococcus neoformans proteins in rodents and humans. Infect Immun 67, 2218-2224.

Cherniak, R., Jones, R.G., and Slodki, M.E. (1988). Type-specific polysaccharides of Cryptococcus

neoformans. n.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella

mesenterica exopolysaccharide. Carbohydr Res 182, 227-239.

Cherniak, R., and Sundstrom, J.B. (1994). Polysaccharide antigens of the capsule of Cryptococcus

neoformans. Infect Immun 62, 1507-1512.

Cianciotto, N.P. (2005). Type II secretion: a protein secretion system for all seasons. Trends

Microbiol 13, 581-588.

Cox, G.M., McDade, H.C., Chen, S.C., Tucker, S.C., Gottfredsson, M., Wright, L.C., Sorrell, T.C.,

Leidich, S.D., Casadevall, A., Ghannoum, M.A., et al. (2001). Extracellular phospholipase activity is

a virulence factor for Cryptococcus neoformans. Mol Microbiol 39, 166-175.

Craig, L., and Li, J. (2008). Type IV pili: paradoxes in form and function. Curr Opin Struct Biol 18,

267-277.

de Gassart, A., Geminard, C., Fevrier, B., Raposo, G., and Vidal, M. (2003). Lipid raft-associated

protein sorting in exosomes. Blood 102, 4336-4344.

132

de Souza Pereira, R., and Geibel, J. (1999). Direct observation of oxidative stress on the cell wall

of Saccharomyces cerevisiae strains with atomic force microscopy. Mol Cell Biochem 201, 17-24.

Del Poeta, M. (2004). Role of phagocytosis in the virulence of Cryptococcus neoformans.

Eukaryot Cell 3, 1067-1075.

Dessauer, A., Rother, U., and Rother, K. (1984). Freeze-thaw activation of the complement attack

phase: I. Separation of two steps in the formation of the active C--56 complex. Acta Pathol

Microbiol Immunol Scand Suppl 284, 75-81.

Diamond, R.D., and Bennett, J.E. (1973). Growth of Cryptococcus neoformans within human

macrophages in vitro. Infect Immun 7, 231-236.

Diamond, R.D., May, J.E., Kane, M.A., Frank, M.M., and Bennett, J.E. (1974). The role of the

classical and alternate complement pathways in host defenses against Cryptococcus neoformans

infection. J Immunol 112, 2260-2270.

Doering, T.L., Nosanchuk, J.D., Roberts, W.K., and Casadevall, A. (1999). Melanin as a potential

cryptococcal defence against microbicidal proteins. Med Mycol 37, 175-181.

Dong, Z.M., and Murphy, J.W. (1997). Cryptococcal polysaccharides bind to CD18 on human

neutrophils. Infect Immun 65, 557-563.

Driessen, A.J., Fekkes, P., and van der Wolk, J.P. (1998). The Sec system. Curr Opin Microbiol 1,

216-222.

Drubin, D.G., and Nelson, W.J. (1996). Origins of cell polarity. Cell 84, 335-344.

Duran, J.M., Anjard, C., Stefan, C., Loomis, W.F., and Malhotra, V. Unconventional secretion of

Acb1 is mediated by autophagosomes. J Cell Biol 188, 527-536.

Eisenman, H.C., Frases, S., Nicola, A.M., Rodrigues, M.L., and Casadevall, A. (2009). Vesicle-

associated melanization in Cryptococcus neoformans. Microbiology 155, 3860-3867.

133

Ellen, A.F., Albers, S.V., Huibers, W., Pitcher, A., Hobel, C.F., Schwarz, H., Folea, M., Schouten, S.,

Boekema, E.J., Poolman, B., et al. (2009). Proteomic analysis of secreted membrane vesicles of

archaeal Sulfolobus species reveals the presence of endosome sorting complex components.

Extremophiles 13, 67-79.

Eng, J.K., McCormack, A.L., Yates, J.R., 3rd (1994). An approach to correlate tandem mass spectral

data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:

976–989.

Esnault, K., el Moudni, B., Bouchara, J.P., Chabasse, D., and Tronchin, G. (1999). Association of a

myosin immunoanalogue with cell envelopes of Aspergillus fumigatus conidia and its

participation in swelling and germination. Infect Immun 67, 1238-1244.

Falkow, S. (1988). Molecular Koch's postulates applied to microbial pathogenicity. Rev Infect Dis

10 Suppl 2, S274-276.

Feldmesser, M., Kress, Y., and Casadevall, A. (2001). Dynamic changes in the morphology of

Cryptococcus neoformans during murine pulmonary infection. Microbiology 147, 2355-2365.

Feldmesser, M., Kress, Y., Novikoff, P., and Casadevall, A. (2000). Cryptococcus neoformans is a

facultative intracellular pathogen in murine pulmonary infection. Infect Immun 68, 4225-4237.

Feldmesser, M., Tucker, S., and Casadevall, A. (2001). Intracellular parasitism of macrophages by

Cryptococcus neoformans. Trends Microbiol 9, 273-278.

Flesch, I.E., Schwamberger, G., and Kaufmann, S.H. (1989). Fungicidal activity of IFN-gamma-

activated macrophages. Extracellular killing of Cryptococcus neoformans. J Immunol 142, 3219-

3224.

Fonseca, F.L., Nohara, L.L., Cordero, R.J., Frases, S., Casadevall, A., Almeida, I.C., Nimrichter, L.,

and Rodrigues, M.L. Immunomodulatory effects of serotype B glucuronoxylomannan from

Cryptococcus gattii correlate with polysaccharide diameter. Infect Immun 78, 3861-3870.

134

Franzot, S.P., Fries, B.C., Cleare, W., and Casadevall, A. (1998). Genetic relationship between

Cryptococcus neoformans var. neoformans strains of serotypes A and D. J Clin Microbiol 36,

2200-2204.

Frases, S., Nimrichter, L., Viana, N.B., Nakouzi, A., and Casadevall, A. (2008). Cryptococcus

neoformans capsular polysaccharide and exopolysaccharide fractions manifest physical,

chemical, and antigenic differences. Eukaryot Cell 7, 319-327.

French, N., Gray, K., Watera, C., Nakiyingi, J., Lugada, E., Moore, M., Lalloo, D., Whitworth, J.A.,

and Gilks, C.F. (2002). Cryptococcal infection in a cohort of HIV-1-infected Ugandan adults. AIDS

16, 1031-1038.

Garcia-Hermoso, D., Janbon, G., and Dromer, F. (1999). Epidemiological evidence for dormant

Cryptococcus neoformans infection. J Clin Microbiol 37, 3204-3209.

Garcia-Rivera, J., Chang, Y.C., Kwon-Chung, K.J., and Casadevall, A. (2004). Cryptococcus

neoformans CAP59 (or Cap59p) is involved in the extracellular trafficking of capsular

glucuronoxylomannan. Eukaryot Cell 3, 385-392.

Ghannoum, M.A. (1998). Extracellular phospholipases as universal virulence factor in pathogenic

fungi. Nippon Ishinkin Gakkai Zasshi 39, 55-59.

Ghannoum, M.A. (2000). Potential role of phospholipases in virulence and fungal pathogenesis.

Clin Microbiol Rev 13, 122-143, table of contents.

Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A.,

Anderson, K., Andre, B., et al. (2002). Functional profiling of the Saccharomyces cerevisiae

genome. Nature 418, 387-391.

Goldman, D.L., Khine, H., Abadi, J., Lindenberg, D.J., Pirofski, L., Niang, R., and Casadevall, A.

(2001). Serologic evidence for Cryptococcus neoformans infection in early childhood. Pediatrics

107, E66.

Gomez, B.L., and Nosanchuk, J.D. (2003). Melanin and fungi. Curr Opin Infect Dis 16, 91-96.

135

Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., and Tannenbaum, S.R.

(1982). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126, 131-

138.

Hauser, A.R., Kang, P.J., and Engel, J.N. (1998). PepA, a secreted protein of Pseudomonas

aeruginosa, is necessary for cytotoxicity and virulence. Mol Microbiol 27, 807-818.

Hoag, K.A., Lipscomb, M.F., Izzo, A.A., and Street, N.E. (1997). IL-12 and IFN-gamma are required

for initiating the protective Th1 response to pulmonary cryptococcosis in resistant C.B-17 mice.

Am J Respir Cell Mol Biol 17, 733-739.

Hoegl, L., Ollert, M., and Korting, H.C. (1996). The role of Candida albicans secreted aspartic

proteinase in the development of candidoses. J Mol Med 74, 135-142.

Horwitz, M.A. (1983). The Legionnaires' disease bacterium (Legionella pneumophila) inhibits

phagosome-lysosome fusion in human monocytes. J Exp Med 158, 2108-2126.

Hu, G., and Kronstad, J.W. A putative P-type ATPase, Apt1, is involved in stress tolerance and

virulence in Cryptococcus neoformans. Eukaryot Cell 9, 74-83.

Hu, G., Steen, B.R., Lian, T., Sham, A.P., Tam, N., Tangen, K.L., and Kronstad, J.W. (2007).

Transcriptional regulation by protein kinase A in Cryptococcus neoformans. PLoS Pathog 3, e42.

Hube, B. (1996). Candida albicans secreted aspartyl proteinases. Curr Top Med Mycol 7, 55-69.

Hurley, J.H., and Emr, S.D. (2006). The ESCRT complexes: structure and mechanism of a

membrane-trafficking network. Annu Rev Biophys Biomol Struct 35, 277-298.

Hyun, T.K., Uddin, M.N., Rim, Y., and Kim, J.Y. Cell-to-cell trafficking of RNA and RNA silencing

through plasmodesmata. Protoplasma.

Ishihama, Y., Oda, Y., Tabata, T., Sato, T., Nagasu, T., Rappsilber, J., and Mann, M. (2005).

Exponentially modified protein abundance index (emPAI) for estimation of absolute protein

amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4,

1265-1272.

136

Jacobson, E.S., and Ikeda, R. (2005). Effect of melanization upon porosity of the cryptococcal cell

wall. Med Mycol 43, 327-333.

Jahn, R., Lang, T., and Sudhof, T.C. (2003). Membrane fusion. Cell 112, 519-533.

Jeffery, C.J. (1999). Moonlighting proteins. Trends Biochem Sci 24, 8-11.

Johnstone, R.M., Adam, M., and Pan, B.T. (1984). The fate of the transferrin receptor during

maturation of sheep reticulocytes in vitro. Can J Biochem Cell Biol 62, 1246-1254.

Kapteyn, J.C., Van Den Ende, H., and Klis, F.M. (1999). The contribution of cell wall proteins to the

organization of the yeast cell wall. Biochim Biophys Acta 1426, 373-383.

Katzmann, D.J., Sarkar, S., Chu, T., Audhya, A., and Emr, S.D. (2004). Multivesicular body sorting:

ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S. Mol Biol

Cell 15, 468-480.

Kawakami, K., Hossain Qureshi, M., Zhang, T., Koguchi, Y., Xie, Q., Kurimoto, M., and Saito, A.

(1999). Interleukin-4 weakens host resistance to pulmonary and disseminated cryptococcal

infection caused by combined treatment with interferon-gamma-inducing cytokines. Cell

Immunol 197, 55-61.

Kawakami, K., Kohno, S., Kadota, J., Tohyama, M., Teruya, K., Kudeken, N., Saito, A., and Hara, K.

(1995). T cell-dependent activation of macrophages and enhancement of their phagocytic activity

in the lungs of mice inoculated with heat-killed Cryptococcus neoformans: involvement of IFN-

gamma and its protective effect against cryptococcal infection. Microbiol Immunol 39, 135-143.

Kinseth, M.A., Anjard, C., Fuller, D., Guizzunti, G., Loomis, W.F., and Malhotra, V. (2007). The

Golgi-associated protein GRASP is required for unconventional protein secretion during

development. Cell 130, 524-534.

Kirchhausen, T. (2000). Three ways to make a vesicle. Nat Rev Mol Cell Biol 1, 187-198.

137

Kleinschek, M.A., Muller, U., Brodie, S.J., Stenzel, W., Kohler, G., Blumenschein, W.M.,

Straubinger, R.K., McClanahan, T., Kastelein, R.A., and Alber, G. (2006). IL-23 enhances the

inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern

distinct from IL-12. J Immunol 176, 1098-1106.

Klis, F.M., Mol, P., Hellingwerf, K., and Brul, S. (2002). Dynamics of cell wall structure in

Saccharomyces cerevisiae. FEMS Microbiol Rev 26, 239-256.

Klutts, J.S., Levery, S.B., and Doering, T.L. (2007). A beta-1,2-xylosyltransferase from Cryptococcus

neoformans defines a new family of glycosyltransferases. J Biol Chem 282, 17890-17899.

Kornfeld, S., and Mellman, I. (1989). The biogenesis of lysosomes. Annu Rev Cell Biol 5, 483-525.

Kozel, T.R., deJong, B.C., Grinsell, M.M., MacGill, R.S., and Wall, K.K. (1998). Characterization of

anticapsular monoclonal antibodies that regulate activation of the complement system by the

Cryptococcus neoformans capsule. Infect Immun 66, 1538-1546.

Kozel, T.R., MacGill, R.S., and Wall, K.K. (1998). Bivalency is required for anticapsular monoclonal

antibodies to optimally suppress activation of the alternative complement pathway by the

Cryptococcus neoformans capsule. Infect Immun 66, 1547-1553.

Kozel, T.R., and Pfrommer, G.S. (1986). Activation of the complement system by Cryptococcus

neoformans leads to binding of iC3b to the yeast. Infect Immun 52, 1-5.

Kuehn, M.J., and Kesty, N.C. (2005). Bacterial outer membrane vesicles and the host-pathogen

interaction. Genes Dev 19, 2645-2655.

Kwon-Chung, K. (1998). Gene disruption to evaluate the role of fungal candidate virulence genes.

Curr Opin Microbiol 1, 381-389.

Leal-Morales, C.A., Bracker, C.E., and Bartnicki-Garcia, S. (1994). Subcellular localization,

abundance and stability of chitin synthetases 1 and 2 from Saccharomyces cerevisiae.

Microbiology 140 ( Pt 9), 2207-2216.

138

Lee, E.Y., Choi, D.Y., Kim, D.K., Kim, J.W., Park, J.O., Kim, S., Kim, S.H., Desiderio, D.M., Kim, Y.K.,

Kim, K.P., et al. (2009). Gram-positive bacteria produce membrane vesicles: proteomics-based

characterization of Staphylococcus aureus-derived membrane vesicles. Proteomics 9, 5425-5436.

Letourneur, F., Gaynor, E.C., Hennecke, S., Demolliere, C., Duden, R., Emr, S.D., Riezman, H., and

Cosson, P. (1994). Coatomer is essential for retrieval of dilysine-tagged proteins to the

endoplasmic reticulum. Cell 79, 1199-1207.

Levin, D.E. (2005). Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol

Rev 69, 262-291.

Levitz, S.M., Dupont, M.P., and Smail, E.H. (1994). Direct activity of human T lymphocytes and

natural killer cells against Cryptococcus neoformans. Infect Immun 62, 194-202.

Levitz, S.M., Nong, S.H., Seetoo, K.F., Harrison, T.S., Speizer, R.A., and Simons, E.R. (1999).

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages. Infect

Immun 67, 885-890.

Levitz, S.M., Tabuni, A., Kozel, T.R., MacGill, R.S., Ingalls, R.R., and Golenbock, D.T. (1997). Binding

of Cryptococcus neoformans to heterologously expressed human complement receptors. Infect

Immun 65, 931-935.

Littman, M.L. (1958). Capsule synthesis by Cryptococcus neoformans. Trans N Y Acad Sci 20, 623-

648.

Liu, H., Sadygov, R.G., and Yates, J.R., 3rd (2004). A model for random sampling and estimation of

relative protein abundance in shotgun proteomics. Anal Chem 76, 4193-4201.

Lupo, P., Chang, Y.C., Kelsall, B.L., Farber, J.M., Pietrella, D., Vecchiarelli, A., Leon, F., and Kwon-

Chung, K.J. (2008). The presence of capsule in Cryptococcus neoformans influences the gene

expression profile in dendritic cells during interaction with the fungus. Infect Immun 76, 1581-

1589.

Ma, H., Croudace, J.E., Lammas, D.A., and May, R.C. (2006). Expulsion of live pathogenic yeast by

macrophages. Curr Biol 16, 2156-2160.

139

Makarova, K.S., Yutin, N., Bell, S.D., and Koonin, E.V. Evolution of diverse cell division and vesicle

formation systems in Archaea. Nat Rev Microbiol 8, 731-741.

Manjithaya, R., Anjard, C., Loomis, W.F., and Subramani, S. Unconventional secretion of Pichia

pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome

formation. J Cell Biol 188, 537-546.

Marzesco, A.M., Janich, P., Wilsch-Brauninger, M., Dubreuil, V., Langenfeld, K., Corbeil, D., and

Huttner, W.B. (2005). Release of extracellular membrane particles carrying the stem cell marker

prominin-1 (CD133) from neural progenitors and other epithelial cells. J Cell Sci 118, 2849-2858.

Mayor, S., and Riezman, H. (2004). Sorting GPI-anchored proteins. Nat Rev Mol Cell Biol 5, 110-

120.

McFadden, D.C., De Jesus, M., and Casadevall, A. (2006). The physical properties of the capsular

polysaccharides from Cryptococcus neoformans suggest features for capsule construction. J Biol

Chem 281, 1868-1875.

McGrath, J.P., and Varshavsky, A. (1989). The yeast STE6 gene encodes a homologue of the

mammalian multidrug resistance P-glycoprotein. Nature 340, 400-404.

Mednick, A.J., Feldmesser, M., Rivera, J., and Casadevall, A. (2003). Neutropenia alters lung

cytokine production in mice and reduces their susceptibility to pulmonary cryptococcosis. Eur J

Immunol 33, 1744-1753.

Milam, J.E., Herring-Palmer, A.C., Pandrangi, R., McDonald, R.A., Huffnagle, G.B., and Toews, G.B.

(2007). Modulation of the pulmonary type 2 T-cell response to Cryptococcus neoformans by

intratracheal delivery of a tumor necrosis factor alpha-expressing adenoviral vector. Infect

Immun 75, 4951-4958.

Miller, M.F., and Mitchell, T.G. (1991). Killing of Cryptococcus neoformans strains by human

neutrophils and monocytes. Infect Immun 59, 24-28.

Monari, C., Bevilacqua, S., Piccioni, M., Pericolini, E., Perito, S., Calvitti, M., Bistoni, F., Kozel, T.R.,

and Vecchiarelli, A. (2009). A microbial polysaccharide reduces the severity of rheumatoid

140

arthritis by influencing Th17 differentiation and proinflammatory cytokines production. J

Immunol 183, 191-200.

Monari, C., Bistoni, F., and Vecchiarelli, A. (2006). Glucuronoxylomannan exhibits potent

immunosuppressive properties. FEMS Yeast Res 6, 537-542.

Mukherjee, J., Nussbaum, G., Scharff, M.D., and Casadevall, A. (1995). Protective and

nonprotective monoclonal antibodies to Cryptococcus neoformans originating from one B cell. J

Exp Med 181, 405-409.

Muller, U., Stenzel, W., Kohler, G., Werner, C., Polte, T., Hansen, G., Schutze, N., Straubinger,

R.K., Blessing, M., McKenzie, A.N., et al. (2007). IL-13 induces disease-promoting type 2

cytokines, alternatively activated macrophages and allergic inflammation during pulmonary

infection of mice with Cryptococcus neoformans. J Immunol 179, 5367-5377.

Nickel, W., and Rabouille, C. (2009). Mechanisms of regulated unconventional protein secretion.

Nat Rev Mol Cell Biol 10, 148-155.

Nimrichter, L., Frases, S., Cinelli, L.P., Viana, N.B., Nakouzi, A., Travassos, L.R., Casadevall, A., and

Rodrigues, M.L. (2007). Self-aggregation of Cryptococcus neoformans capsular

glucuronoxylomannan is dependent on divalent cations. Eukaryot Cell 6, 1400-1410.

Nimrichter, L., Rodrigues, M.L., Rodrigues, E.G., and Travassos, L.R. (2005). The multitude of

targets for the immune system and drug therapy in the fungal cell wall. Microbes Infect 7, 789-

798.

Nosanchuk, J.D., Steenbergen, J.N., Shi, L., Deepe, G.S., Jr., and Casadevall, A. (2003). Antibodies

to a cell surface histone-like protein protect against Histoplasma capsulatum. J Clin Invest 112,

1164-1175.

Novick, P., Field, C., and Schekman, R. (1980). Identification of 23 complementation groups

required for post-translational events in the yeast secretory pathway. Cell 21, 205-215.

141

Novick, P., and Schekman, R. (1979). Secretion and cell-surface growth are blocked in a

temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 76, 1858-

1862.

Novick, P., and Schekman, R. (1983). Export of major cell surface proteins is blocked in yeast

secretory mutants. J Cell Biol 96, 541-547.

Oberholzer, U., Marcil, A., Leberer, E., Thomas, D.Y., and Whiteway, M. (2002). Myosin I is

required for hypha formation in Candida albicans. Eukaryot Cell 1, 213-228.

Ortiz, D., and Novick, P.J. (2006). Ypt32p regulates the translocation of Chs3p from an internal

pool to the plasma membrane. Eur J Cell Biol 85, 107-116.

Osterholzer, J.J., Milam, J.E., Chen, G.H., Toews, G.B., Huffnagle, G.B., and Olszewski, M.A. (2009).

Role of dendritic cells and alveolar macrophages in regulating early host defense against

pulmonary infection with Cryptococcus neoformans. Infect Immun 77, 3749-3758.

Palade, G. (1975). Intracellular aspects of the process of protein synthesis. Science 189, 347-358.

Pan, B.T., Teng, K., Wu, C., Adam, M., and Johnstone, R.M. (1985). Electron microscopic evidence

for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol

101, 942-948.

Panepinto, J., Komperda, K., Frases, S., Park, Y.D., Djordjevic, J.T., Casadevall, A., and Williamson,

P.R. (2009). Sec6-dependent sorting of fungal extracellular exosomes and laccase of

Cryptococcus neoformans. Mol Microbiol 71, 1165-1176.

Park, B.J., Wannemuehler, K.A., Marston, B.J., Govender, N., Pappas, P.G., and Chiller, T.M.

(2009). Estimation of the current global burden of cryptococcal meningitis among persons living

with HIV/AIDS. AIDS 23, 525-530.

Pierini, L.M., and Doering, T.L. (2001). Spatial and temporal sequence of capsule construction in

Cryptococcus neoformans. Mol Microbiol 41, 105-115.

142

Pietrella, D., Corbucci, C., Perito, S., Bistoni, G., and Vecchiarelli, A. (2005). Mannoproteins from

Cryptococcus neoformans promote dendritic cell maturation and activation. Infect Immun 73,

820-827.

Poisson, G., Chauve, C., Chen, X., and Bergeron, A. (2007). FragAnchor: a large-scale predictor of

glycosylphosphatidylinositol anchors in eukaryote protein sequences by qualitative scoring.

Genomics Proteomics Bioinformatics 5, 121-130.

Polacheck, I. (1991). The discovery of melanin production in Cryptococcus neoformans and its

impact on diagnosis and the study of virulence. Zentralbl Bakteriol 276, 120-123.

Ponnambalam, S., and Baldwin, S.A. (2003). Constitutive protein secretion from the trans-Golgi

network to the plasma membrane. Mol Membr Biol 20, 129-139.

Qu, Y., Franchi, L., Nunez, G., and Dubyak, G.R. (2007). Nonclassical IL-1 beta secretion stimulated

by P2X7 receptors is dependent on inflammasome activation and correlated with exosome

release in murine macrophages. J Immunol 179, 1913-1925.

Raposo, G., Nijman, H.W., Stoorvogel, W., Liejendekker, R., Harding, C.V., Melief, C.J., and Geuze,

H.J. (1996). B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183, 1161-1172.

Rawat, D.S., Upreti, H.B., and Das, S.K. (1984). Lipid composition of Cryptococcus neoformans.

Microbiologica 7, 299-307.

Regente, M., Corti-Monzon, G., Maldonado, A.M., Pinedo, M., Jorrin, J., and de la Canal, L.

(2009). Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome

marker proteins. FEBS Lett 583, 3363-3366.

Rhodes, J.C. (1985). Contribution of complement component C5 to the pathogenesis of

experimental murine cryptococcosis. Sabouraudia 23, 225-234.

Rhodes, J.C., Wicker, L.S., and Urba, W.J. (1980). Genetic control of susceptibility to Cryptococcus

neoformans in mice. Infect Immun 29, 494-499.

Rice, L.M., and Brunger, A.T. (1999). Crystal structure of the vesicular transport protein Sec17:

implications for SNAP function in SNARE complex disassembly. Mol Cell 4, 85-95.

143

Rittershaus, P.C., Kechichian, T.B., Allegood, J.C., Merrill, A.H., Jr., Hennig, M., Luberto, C., and

Del Poeta, M. (2006). Glucosylceramide synthase is an essential regulator of pathogenicity of

Cryptococcus neoformans. J Clin Invest 116, 1651-1659.

Rivera, J., Cordero, R.J., Nakouzi, A.S., Frases, S., Nicola, A., and Casadevall, A. Bacillus anthracis

produces membrane-derived vesicles containing biologically active toxins. Proc Natl Acad Sci U S

A 107, 19002-19007.

Rivera, J., Feldmesser, M., Cammer, M., and Casadevall, A. (1998). Organ-dependent variation of

capsule thickness in Cryptococcus neoformans during experimental murine infection. Infect

Immun 66, 5027-5030.

Rodrigues, M.L., Nakayasu, E.S., Oliveira, D.L., Nimrichter, L., Nosanchuk, J.D., Almeida, I.C., and

Casadevall, A. (2008). Extracellular vesicles produced by Cryptococcus neoformans contain

protein components associated with virulence. Eukaryot Cell 7, 58-67.

Rodrigues, M.L., Shi, L., Barreto-Bergter, E., Nimrichter, L., Farias, S.E., Rodrigues, E.G., Travassos,

L.R., and Nosanchuk, J.D. (2007). Monoclonal antibody to fungal glucosylceramide protects mice

against lethal Cryptococcus neoformans infection. Clin Vaccine Immunol 14, 1372-1376.

Rodrigues, M.L., Travassos, L.R., Miranda, K.R., Franzen, A.J., Rozental, S., de Souza, W., Alviano,

C.S., and Barreto-Bergter, E. (2000). Human antibodies against a purified glucosylceramide from

Cryptococcus neoformans inhibit cell budding and fungal growth. Infect Immun 68, 7049-7060.

Rothman, J.E. (1994). Mechanisms of intracellular protein transport. Nature 372, 55-63.

Sakaguchi, N., Baba, T., Fukuzawa, M., and Ohno, S. (1993). Ultrastructural study of Cryptococcus

neoformans by quick-freezing and deep-etching method. Mycopathologia 121, 133-141.

Schekman, R. (2002). Lasker Basic Medical Research Award. SEC mutants and the secretory

apparatus. Nat Med 8, 1055-1058.

144

Schekman, R., Esmon, B., Ferro-Novick, S., Field, C., and Novick, P. (1983). Yeast secretory

mutants: isolation and characterization. Methods Enzymol 96, 802-815.

Schorey, J.S., and Bhatnagar, S. (2008). Exosome function: from tumor immunology to pathogen

biology. Traffic 9, 871-881.

Segev, N. (2001). Ypt/rab gtpases: regulators of protein trafficking. Sci STKE 2001, re11.

Shapiro, S., Beenhouwer, D.O., Feldmesser, M., Taborda, C., Carroll, M.C., Casadevall, A., and

Scharff, M.D. (2002). Immunoglobulin G monoclonal antibodies to Cryptococcus neoformans

protect mice deficient in complement component C3. Infect Immun 70, 2598-2604.

Shoham, S., Huang, C., Chen, J.M., Golenbock, D.T., and Levitz, S.M. (2001). Toll-like receptor 4

mediates intracellular signaling without TNF-alpha release in response to Cryptococcus

neoformans polysaccharide capsule. J Immunol 166, 4620-4626.

Shorter, J., Watson, R., Giannakou, M.E., Clarke, M., Warren, G., and Barr, F.A. (1999). GRASP55,

a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free

system. EMBO J 18, 4949-4960.

Simons, M., and Raposo, G. (2009). Exosomes--vesicular carriers for intercellular communication.

Curr Opin Cell Biol 21, 575-581.

Singh, N., Alexander, B.D., Lortholary, O., Dromer, F., Gupta, K.L., John, G.T., del Busto, R.,

Klintmalm, G.B., Somani, J., Lyon, G.M., et al. (2008). Pulmonary cryptococcosis in solid organ

transplant recipients: clinical relevance of serum cryptococcal antigen. Clin Infect Dis 46, e12-18.

Soler, N., Marguet, E., Verbavatz, J.M., and Forterre, P. (2008). Virus-like vesicles and

extracellular DNA produced by hyperthermophilic archaea of the order Thermococcales. Res

Microbiol 159, 390-399.

Sommer, U., Liu, H., and Doering, T.L. (2003). An alpha-1,3-mannosyltransferase of Cryptococcus

neoformans. J Biol Chem 278, 47724-47730.

Southwick, F.S., and Purich, D.L. (1996). Intracellular pathogenesis of listeriosis. N Engl J Med 334,

770-776.

145

Steenbergen, J.N., and Casadevall, A. (2003). The origin and maintenance of virulence for the

human pathogenic fungus Cryptococcus neoformans. Microbes Infect 5, 667-675.

Stephens, D.J., and Pepperkok, R. (2001). Illuminating the secretory pathway: when do we need

vesicles? J Cell Sci 114, 1053-1059.

Strasser, J.E., Newman, S.L., Ciraolo, G.M., Morris, R.E., Howell, M.L., and Dean, G.E. (1999).

Regulation of the macrophage vacuolar ATPase and phagosome-lysosome fusion by Histoplasma

capsulatum. J Immunol 162, 6148-6154.

Sturgill-Koszycki, S., Schlesinger, P.H., Chakraborty, P., Haddix, P.L., Collins, H.L., Fok, A.K., Allen,

R.D., Gluck, S.L., Heuser, J., and Russell, D.G. (1994). Lack of acidification in Mycobacterium

phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263, 678-681.

Subra, C., Laulagnier, K., Perret, B., and Record, M. (2007). Exosome lipidomics unravels lipid

sorting at the level of multivesicular bodies. Biochimie 89, 205-212.

Sutton, R.B., Fasshauer, D., Jahn, R., and Brunger, A.T. (1998). Crystal structure of a SNARE

complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395, 347-353.

Tokuyasu, K.T. (1973). A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol

57, 551-565.

Tu, J., Vallier, L.G., and Carlson, M. (1993). Molecular and genetic analysis of the SNF7 gene in

Saccharomyces cerevisiae. Genetics 135, 17-23.

Tucker, S.C., and Casadevall, A. (2002). Replication of Cryptococcus neoformans in macrophages

is accompanied by phagosomal permeabilization and accumulation of vesicles containing

polysaccharide in the cytoplasm. Proc Natl Acad Sci U S A 99, 3165-3170.

Vaishnav, V.V., Bacon, B.E., O'Neill, M., and Cherniak, R. (1998). Structural characterization of the

galactoxylomannan of Cryptococcus neoformans Cap67. Carbohydr Res 306, 315-330.

146

Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J.J., and Lotvall, J.O. (2007). Exosome-

mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between

cells. Nat Cell Biol 9, 654-659.

Valdivia, R.H., and Schekman, R. (2003). The yeasts Rho1p and Pkc1p regulate the transport of

chitin synthase III (Chs3p) from internal stores to the plasma membrane. Proc Natl Acad Sci U S A

100, 10287-10292.

Vecchiarelli, A. (2000). Immunoregulation by capsular components of Cryptococcus neoformans.

Med Mycol 38, 407-417.

Villena, S.N., Pinheiro, R.O., Pinheiro, C.S., Nunes, M.P., Takiya, C.M., DosReis, G.A., Previato, J.O.,

Mendonca-Previato, L., and Freire-de-Lima, C.G. (2008). Capsular polysaccharides

galactoxylomannan and glucuronoxylomannan from Cryptococcus neoformans induce

macrophage apoptosis mediated by Fas ligand. Cell Microbiol 10, 1274-1285.

Voelz, K., and May, R.C. Cryptococcal interactions with the host immune system. Eukaryot Cell 9,

835-846.

Waterman, S.R., Hacham, M., Panepinto, J., Hu, G., Shin, S., and Williamson, P.R. (2007). Cell wall

targeting of laccase of Cryptococcus neoformans during infection of mice. Infect Immun 75, 714-

722.

Waters, M.G., Serafini, T., and Rothman, J.E. (1991). 'Coatomer': a cytosolic protein complex

containing subunits of non-clathrin-coated Golgi transport vesicles. Nature 349, 248-251.

Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T.H.,

and Rothman, J.E. (1998). SNAREpins: minimal machinery for membrane fusion. Cell 92, 759-772.

Williamson, P.R. (1997). Laccase and melanin in the pathogenesis of Cryptococcus neoformans.

Front Biosci 2, e99-107.

Winzeler, E.A., Shoemaker, D.D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R.,

Benito, R., Boeke, J.D., Bussey, H., et al. (1999). Functional characterization of the S. cerevisiae

genome by gene deletion and parallel analysis. Science 285, 901-906.

147

Wolfers, J., Lozier, A., Raposo, G., Regnault, A., Thery, C., Masurier, C., Flament, C., Pouzieux, S.,

Faure, F., Tursz, T., et al. (2001). Tumor-derived exosomes are a source of shared tumor rejection

antigens for CTL cross-priming. Nat Med 7, 297-303.

Wuestehube, L.J., Duden, R., Eun, A., Hamamoto, S., Korn, P., Ram, R., and Schekman, R. (1996).

New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the

endoplasmic reticulum to the Golgi complex. Genetics 142, 393-406.

Yamaguchi, M., Ohkusu, M., Sameshima, M., and Kawamoto, S. (2005). Safe specimen

preparation for electron microscopy of pathogenic fungi by freeze-substitution after

glutaraldehyde fixation. Nippon Ishinkin Gakkai Zasshi 46, 187-192.

Yeo, S.C., Xu, L., Ren, J., Boulton, V.J., Wagle, M.D., Liu, C., Ren, G., Wong, P., Zahn, R., Sasajala,

P., et al. (2003). Vps20p and Vta1p interact with Vps4p and function in multivesicular body

sorting and endosomal transport in Saccharomyces cerevisiae. J Cell Sci 116, 3957-3970.

Yoneda, A., and Doering, T.L. (2006). A eukaryotic capsular polysaccharide is synthesized

intracellularly and secreted via exocytosis. Mol Biol Cell 17, 5131-5140.

Yonezawa, H., Osaki, T., Kurata, S., Fukuda, M., Kawakami, H., Ochiai, K., Hanawa, T., and Kamiya,

S. (2009). Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm

formation. BMC Microbiol 9, 197.

Zaragoza, O., and Casadevall, A. (2004). Experimental modulation of capsule size in Cryptococcus

neoformans. Biol Proced Online 6, 10-15.

Zaragoza, O., Chrisman, C.J., Castelli, M.V., Frases, S., Cuenca-Estrella, M., Rodriguez-Tudela, J.L.,

and Casadevall, A. (2008). Capsule enlargement in Cryptococcus neoformans confers resistance

to oxidative stress suggesting a mechanism for intracellular survival. Cell Microbiol 10, 2043-

2057.

Zaragoza, O., Rodrigues, M.L., De Jesus, M., Frases, S., Dadachova, E., and Casadevall, A. (2009).

The capsule of the fungal pathogen Cryptococcus neoformans. Adv Appl Microbiol 68, 133-216.

148

Zaragoza, O., Telzak, A., Bryan, R.A., Dadachova, E., and Casadevall, A. (2006). The polysaccharide

capsule of the pathogenic fungus Cryptococcus neoformans enlarges by distal growth and is

rearranged during budding. Mol Microbiol 59, 67-83.

Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., Flament, C., Tenza, D., Ricciardi-Castagnoli, P.,

Raposo, G., and Amigorena, S. (1998). Eradication of established murine tumors using a novel

cell-free vaccine: dendritic cell-derived exosomes. Nat Med 4, 594-600.

149

Anexo 5: Esta seção reune os demais artigos nos quais participei como autora

no decorrer do meu doutorado.

EUKARYOTIC CELL, Jan. 2008, p. 58–67 Vol. 7, No. 1

1535-9778/08/$08.00ϩ0 doi:10.1128/EC.00370-07

Extracellular Vesicles Produced by Cryptococcus neoformans Contain

Protein Components Associated with Virulence

ᰔ

†

Marcio L. Rodrigues,

* Ernesto S. Nakayasu,

Debora L. Oliveira,

Leonardo Nimrichter,

Joshua D. Nosanchuk,

3,4

Igor C. Almeida,

and Arturo Casadevall

3,4

Laborato´rio de Estudos Integrados em Bioquı´mica Microbiana, Departamento de Microbiologia Geral, Instituto de

Microbiologia Professor Paulo de Go´es, Universidade Federal do Rio de Janeiro, CEP 21941590 Rio de Janeiro, Brazil

;

Department of Biological Sciences, The Border Biomedical Research Center, University of Texas at El Paso,

El Paso, Texas 79968-0519

; and Department of Microbiology and Immunology

and Division of

Infectious Diseases,

Department of Medicine, Albert Einstein College of Medicine,

1300 Morris Park Ave., Bronx, New York 10461

Received 8 October 2007/Accepted 6 November 2007

Cryptococcus neoformans produces vesicles containing its major virulence factor, the capsular polysaccharide

glucuronoxylomannan (GXM). These vesicles cross the cell wall to reach the extracellular space, where the

polysaccharide is supposedly used for capsule growth or delivered into host tissues. In the present study, we

characterized vesicle morphology and protein composition by a combination of techniques including electron

microscopy, proteomics, enzymatic activity, and serological reactivity. Secretory vesicles in C. neoformans

appear to be correlated with exosome-like compartments derived from multivesicular bodies. Extracellular

vesicles manifested various sizes and morphologies, including electron-lucid membrane bodies and electron-

dense vesicles. Seventy-six proteins were identiﬁed by proteomic analysis, including several related to virulence

and protection against oxidative stress. Biochemical tests indicated laccase and urease activities in vesicles. In

addition, different vesicle proteins were recognized by sera from patients with cryptococcosis. These results

reveal an efﬁcient and general mechanism of secretion of pathogenesis-related molecules in C. neoformans,

suggesting that extracellular vesicles function as “virulence bags” that deliver a concentrated payload of fungal

products to host effector cells and tissues.

Cryptococcus neoformans is the causative agent of cryptococ-

cosis and a major pathogen for immunosuppressed individuals

(39). Cryptococcosis is rampant in Africa and Asia in associa-

tion with human immunodeﬁciency virus infection, where it is

associated with high morbidity and mortality (5). C. neofor-

mans provides a unique model in cell biology studies because

it is the only eukaryotic pathogen with a polysaccharide cap-

sule, a structure that is essential for virulence (26, 32, 39). The

major capsular polysaccharide, glucuronoxylomannan (GXM),

is released extracellularly during infection and induces a num-

ber of deleterious effects to the host, including interference

with phagocytosis, inhibition of leukocyte migration to infected

tissues, and modulation of cytokine production (reviewed in

reference 32). It also represents a potential component for

vaccines (13) and is the target of potentially therapeutic anti-

bodies (7).

The mechanisms of secretion of macromolecules by fungal

cells have not been well elucidated. Fungal cells are encased in

a rigid, pore-containing cell wall consisting of polysaccharides,

proteins, and pigments (14, 35). Different studies demonstrate

that structures with molecular masses higher than 1,000 kDa

can cross the cell wall and reach the extracellular milieu (26,

27, 47). GXM, for instance, has an average molecular mass

ranging from 1.7 ϫ 10

to 7 ϫ 10

Da (27). Several studies by

our group and others indicate that C. neoformans synthesizes

GXM intracellularly and then transports the polysaccharide to

the extracellular space for assembly into a capsule (17, 20, 45,

54, 55). The mechanism by which capsular polysaccharide is

synthesized inside the cell and exported to the extracellular

environment for capsule assembly and release is a central ques-

tion in cryptococcal cell biology.

We have recently described how GXM-containing vesicles

accumulate in supernatants of C. neoformans cultures (45).

These extracellular vesicles contain bilayered membranes en-

riched with key fungal lipids, such as glucosylceramide and

sterols. This observation led to the proposal that extracellular

export of GXM is accomplished by vesicular transport (45).

The existence of a vesicular transport mechanism raises the

possibility that other fungal molecules could be released using

the same cellular apparatus. Indeed, a secretion mutant of C.

neoformans that accumulates secretory vesicles in the cyto-

plasm has severe defects in protein secretion (54). We there-

fore hypothesized that vesicular extracellular secretion in C.

neoformans is a general mechanism used for the trans-cell-wall

transport of protein, lipid, and carbohydrate components to

the extracellular environment.

In the present work, we used microscopic, serological, bio-

chemical, and proteomic approaches to analyze the extracel-

lular vesicles of C. neoformans. Electron microscopy suggests

* Corresponding author. Mailing address for M. L. Rodrigues: In-

stituto de Microbiologia Professor Paulo de Go´es, Universidade Fed-

eral do Rio de Janeiro, 21941590 Rio de Janeiro, Brazil. Phone: 55 21

2562 6740. Fax: 55 21 25608344. E-mail: [email protected].

Mailing address for A. Casadevall: Department of Microbiology and

Immunology, Albert Einstein College of Medicine, 1300 Morris Park

Avenue, Bronx, NY 10461. Phone: (718) 430-2215. Fax: (718) 430-8968.

E-mail: [email protected].

† Supplemental material for this article may be found at http://ec

.asm.org/.

ᰔ

Published ahead of print on 26 November 2007.

that vesicle secretion derives from the trafﬁc of multivesicle-

like compartments to the cell surface. The results indicate that

C. neoformans extracellular vesicles represent a heterogeneous

population of “virulence bags” containing numerous molecules

associated with fungal survival and host pathogenicity.

MATERIALS AND METHODS

Strains. The C. neoformans isolates used in this study included strains H99

(serotype A, wild type), Cap 67 (derived from serotype D and lacking a GXM

capsule), and 2E-TU and 2E-TUC (serotype D, LAC1 gene deletion and recon-

stituted laccase mutants) (46). Yeast cells were cultivated in a minimal medium

composed of dextrose (15 mM), MgSO

(10 mM), KH

(29.4 mM), glycine

(13 mM), and thiamine-HCl (3 ␮M). Fungal cells were cultivated for 48 h at

30°C, with continuous shaking. Strain Cap 67 was used for electron microscopy.

Strains 2E-TU and 2E-TUC were used for laccase assays. Strain H99 was used

for all other experiments. Proteomic analyses were performed using strains Cap

67, H99, and 2E-TUC.

Vesicle puriﬁcation. Isolation of extracellular vesicles was done using the

protocol described by Rodrigues et al. (45). Brieﬂy, cell-free culture supernatants

were obtained by sequential centrifugation at 4,000 and 15,000 ϫ g (15 min, 4°C).

These supernatants contained vesicles and were concentrated by approximately

20-fold using an Amicon ultraﬁltration system (cutoff of 100 kDa). The concen-

trate was again centrifuged at 4,000 and 15,000 ϫ g (15 min, 4°C) and then at

100,000 ϫ g for1hat4°C. The supernatants were discarded, and pellets were

washed by ﬁve sequential suspension and centrifugation steps, each consisting of

100,000 ϫ g for1hat4°Cwith 0.1 M Tris-buffered saline. To remove extrave-

sicular GXM contamination, vesicles were subjected to passage through a col-

umn packed with cyanogen bromide-activated Sepharose coupled to a monoclo-

nal antibody to GXM, as described previously (45). Fractions that were not

bound to the monclonal antibody-containing column were again centrifuged at

100,000 ϫ g. The resulting pellets were then suspended in ﬁxative solution for

electron microscopy analysis or prepared for biochemical, proteomic, and West-

ern blotting analyses, as described below.

TEM. For transmission electron microscopy (TEM), C. neoformans cells were

grown in minimal medium, serially washed in phosphate-buffered saline (PBS),

and ﬁxed for1hin0.1Msodium cacodylate buffer (pH 7.2) containing 4%

paraformaldehyde and 2% glutaraldehyde. Cells were then inﬁltrated for 2 h in

a solution containing 25% polyvinylpyrrolidone and 2.1 M sucrose and then

rapidly frozen by immersion in liquid nitrogen. They were transferred to a

cryo-ultramicrotome (Ultracut; Reichert), and cryosections were obtained in a

temperature range of Ϫ70 to Ϫ90°C. The material was collected with a sucrose

loop and transferred to Formvar-carbon-coated grids. Specimens were observed

in a Zeiss 900 transmission electron microscope operating at 80 kV.

Pellets obtained after centrifugation of cell-free supernatants at 100,000 ϫ g

were ﬁxed with 2% glutaraldehyde in 0.1 M cacodylate at room temperature for

2 h and then incubated overnight in 4% formaldehyde, 1% glutaraldehyde, and

0.1% PBS. The samples were incubated for 90 min in 2% osmium, serially

dehydrated in ethanol, and embedded in Spurrs epoxy resin. Thin sections were

obtained on a Reichart Ultracut UCT and stained with 0.5% uranyl acetate and

0.5% lead citrate. Samples were observed in a JEOL 1200EX transmission

electron microscope operating at 80 kV.

Biochemical detection of enzymatic activities. Laccase and urease activity in

vesicle preparations was assayed spectrophotometrically. Acid phosphatase, a

cryptococcal protein that mediates adhesion of cryptococci to epithelial cells and

whose secretion has been associated with vesicle production (8, 54), was also

assayed. Pellets obtained after centrifugation at 100,000 ϫ g were suspended in

PBS and serially diluted in media appropriate for the reactions catalyzed by

laccase, urease, or acid phosphatase. The laccase reaction medium corresponded

to 0.2% (10 mM)

L-DOPA in PBS, while the medium for urease activity con-

tained 4% urea, 0.02% yeast extract, 0.002% phenol red, 0.273% KH

, and

0.285% Na

HPO-

. For phosphatase determination, the reaction medium con

sisted of acetate buffer (pH 5.0) supplemented with 5 mg/ml p-nitrophenyl

phosphate. Vesicle suspensions were incubated overnight at room temperature

and protected from the light. Reactions were quantiﬁed by reading at 450

(laccase), 405 (phosphatase), or 540 (urease) nm with a Multiscan mass spec-

trometer (MS) (Labsystem, Helsinki, Finland). The amount of vesicles in each

system was assumed to be related to the protein concentration in each vesicular

suspension. The protein concentration at the starting dilution in each system

corresponded to 0.3 ␮g/ml. Enzymatic assays were repeated at least three times,

producing similar results.

Western blot analysis. Vesicles were suspended in loading buffer (1% so-

dium dodecyl sulfate [SDS], 10% glycerol, 10 mM Tris-Cl [pH 6.8], 1 mM

2-mercaptoethanol, and 0.05 mg/ml bromophenol blue) and a ﬁnal amount of

proteins corresponding to 8 ␮g was loaded onto 12% SDS-polyacrylamide gel

electrophoresis (PAGE) gel. Separated proteins were transferred to nitrocel-

lulose membranes, which were sequentially blocked in PBS containing 1%

bovine serum albumin and incubated for1hatroom temperature with pooled

sera from 10 individuals diagnosed with cryptococcosis based on positive

serum reactivity in latex agglutination tests for cryptococcal GXM. Alterna-

tively, the membranes were incubated with pooled normal human sera from

healthy volunteers with no previous diagnosis of any systemic mycosis. Pooled

sera were used at a 1:100 dilution in PBS-bovine serum albumin. After

extensive washing, the membranes were incubated in the presence of a per-

oxidase-labeled anti-human immunoglobulin antibody followed by immuno-

detection by chemiluminescence (Pierce). To exclude the possibility of non-

speciﬁc recognition of samples by secondary antibodies, samples were

incubated directly with the peroxidase-labeled antihuman antibody, produc-

ing negative results (not shown). Serological analyses were repeated twice,

with similar results.

Protein identiﬁcation by liquid chromatography-tandem mass spectrometry.

Puriﬁed vesicles were suspended in 400 mM NH

HCO

(40 ␮l) containing 8 M

urea, and 50 mM dithiothreitol (10 ␮l) was then added for reduction of disulﬁde

bonds. After incubation at 50°C for 15 min, the cysteine residues were alkyl-

ated through the addition of 100 mM iodoacetamide (10 ␮l), followed by

incubation for 15 min at room temperature under protection from the light.

The ﬁnal concentration of urea was then adjusted to1Mbytheaddition of

high-performance liquid chromatography (HPLC)-grade water (Sigma). The

mixture was supplemented with 4 ␮g sequencing-grade trypsin (Promega) and

digested overnight at 37°C. The resulting sample was puriﬁed in reverse-

phase ZipTip columns (POROS R2 50; Applied Biosystems) as described by

Jurado et al. (24). Released peptides were then fractionated on a strong

cation-exchange ZipTip column (POROS HS 50 resin; Applied Biosystems),

preequilibrated with 25% acetonitrile (ACN)–0.5% formic acid (FA). After

loading the peptide mixture, the column was washed with 25% ACN–0.5%

FA and the peptides were eluted with the same solution supplemented with

NaCl concentrations ranging from 0 to 500 mM. Each fraction was dried in a

vacuum centrifuge (Eppendorf) and again puriﬁed by reverse-phase chroma-

tography in POROS R2 50 ZipTip columns. Fractions were ﬁnally suspended

in 0.05% triﬂuoracetic acid (30 ␮l). An 8-␮l aliquot of each fraction was

loaded into a C

trap column (1 ␮lC

; OPTI-PAK). The separation was

performed on a capillary reverse-phase column (Acclaim; LC Packings [3 ␮m

,75␮m by 25 cm]) connected to a nanoHPLC system (nanoLC 1D Plus;

Eksigent). Peptides were eluted with increasing concentrations of ACN (0 to

40%) in 0.1% FA during 100 min and directly analyzed in an electrospray-

linear ion trap MS equipped with a nanospray source (LTQ XL; Thermo

Fisher). MS spectra were collected in centroid mode at the 400 to 1,700 m/z

range, and the ﬁve most abundant ions were subjected twice to collision-

induced dissociation with 35% normalized collision energy, before being

dynamically excluded for 120 s.

All tandem MS spectra from peptides with 600 to 4,000 Da, more than 100 counts,

and at least 15 fragments were converted into DTA ﬁles using Bioworks v.3.3.1

(Thermo). The DTA ﬁles were submitted to a database search using TurboSequest

(15), available in Bioworks, and the C. neoformans protein database, available at

www.broad.mit.edu/annotation/fungi/cryptococcus_neoformans. Common contami-

nant sequences (retrieved from GenBank at http://www.ncbi.nlm.nih.gov/ and the

International Protein Index at http://www.ebi.ac.uk/IPI) and 100,000 randomly gen-

erated sequences were used to supplement the C. neoformans database. The data-

base search parameters included (i) trypsin cleavage in both peptide termini with

one missed cleavage site allowed; (ii) carbamidomethylation of cysteine residues as

a ﬁxed modiﬁcation; (iii) oxidation of methionine residues as a variable modiﬁcation;

and (iv) 2.0 and 1.0 Da for peptide and fragment mass tolerance, respectively. To

ensure the quality of protein identiﬁcation, the false-positive rate (FPR) was esti-

mated using the TurboSequest output and the following formula: FPR ϭ number of

proteins matching random sequences/total number of proteins.

The FPR was calculated after applying the following ﬁlters in Bioworks:

distinct peptides (for exclusion of redundant hits); DCn, Ն0.1; protein proba-

bility, Յ1 ϫ 10

Ϫ3

; and Xcorr, Ն1.5, 2.2, and 2.7 for singly, doubly, and triply

charged peptides, respectively. When necessary, protein consensus scores were

also applied to limit the number of false-positive hits. All data sets showed an

FPR lower than 3.2%.

VOL. 7, 2008 C. NEOFORMANS VESICLES AND VIRULENCE PROTEINS 59

RESULTS

The extracellular vesicles produced by C. neoformans consist

of a complex population. The individual analysis of micro-

graphs of 419 secreted vesicles produced by C. neoformans

revealed considerable morphological variation. Using morpho-

logical criteria, we identiﬁed four main vesicle groups (Fig. 1).

The groups included electron-dense and electron-lucid vesi-

cles, vesicular structures with membrane-associated electron-

dense regions, and vesicles containing hyper-dense structures

resembling a dark pigment.

Pathogenesis-related molecules are present in the extracel-

lular vesicles. The microscopic analysis illustrated in Fig. 1

suggested that pigment synthesis could occur in vesicular struc-

tures. We therefore evaluated whether laccase, a cryptococcal

enzyme characterized as a virulence factor responsible for mel-

anin synthesis (46), was present in vesicular preparations. Lac-

case activity was detected in vesicle preparations from wild-

type cells (Fig. 2A). Enzyme activity was absent in vesicles from

a C. neoformans mutant lacking the ability to produce LAC1,

while vesicular structures from a laccase-reconstituted strain

had enzyme activity similar to wild-type cells. These results,

combined with a recent report by our group (45), indicated

that extracellular vesicles in C. neoformans contain at least

three virulence determinants: GXM (reviewed in reference

32), glucosylceramides (44), and laccase (46). We therefore

evaluated whether other molecules related to pathogenesis

were present in the C. neoformans vesicular structures.

We examined the activities of urease, a well-described ex-

tracellular virulence factor of C. neoformans (11), and acid

phosphatase, an adhesion-related molecule (8) suggested to be

released to the extracellular space in secretory vesicles (54).

Dose-dependent activities of both enzymes were observed

when the extracellular vesicles were incubated with urease and

phosphatase substrates (Fig. 2B and C).

Vesicle proteins are recognized by sera from cryptococcosis

patients. Vesicle proteins were separated by SDS-PAGE and

analyzed by reactivity with human sera by immunoblotting

(Fig. 3). No signiﬁcant reactivity was observed using sera from

normal individuals. However, when the vesicle sample was

probed with a pool of sera from cryptococcosis patients, seven

major bands were observed, with relative molecular masses

corresponding to 131, 101, 67, 48, 38, 27, and 19 kDa. Diffuse

FIG. 1. Electron microscopic appearance (left panels) and preva-

lence (right panels) of the four major vesicle morphological groups

observed in preparations of extracellular vesicles from C. neoformans.

The total population analyzed consisted of 419 different vesicles. Scale

bars, 200 nm.

FIG. 2. Laccase (A), urease (B), and phosphatase (C) activities are associated with extracellular vesicles in C. neoformans. (A). Vesicles puriﬁed

from culture supernatants of strains H99 and the serotype D laccase mutants 2E-TUC (complemented strain) and 2E-TU (LAC1 deletion strain)

were incubated in the presence of L-DOPA and analyzed spectrophotometrically. Vesicles puriﬁed from the supernatants of strain H99 were also

incubated in urease (B) and phosphatase (C) reaction media, followed by spectrophotometric determination of enzyme activity.

FIG. 3. Vesicle-associated proteins are recognized by sera from

cryptococcosis patients. Vesicle-associated proteins (a and b) were

separated by SDS-PAGE and incubated with pooled sera from healthy

individuals (a) or cryptococcosis patients (b). Molecular masses for

standard (left values) or vesicle (right values) proteins are indicated.

60 RODRIGUES ET AL. E

UKARYOT.CELL

TABLE 1. Protein components of C. neoformans extracellular vesicles

Hit no. Accession no./identiﬁcation Function (reference) Strain(s)

Pathogenesis/immune

response

1 CNAG_01727.1/heat shock 70-kDa protein 2*

(644 aa, 69.53 kDa)

Chaperone Cap67, 2E-TUC, H99

2 CNAG_03891.1/60-kDa chaperonin* (582 aa,

61.36 kDa)

Chaperone Cap67

3 CNAG_06150.1/heat shock protein 90* (700 aa,

79.15 kDa)

Chaperone Cap67

4 CNAG_00334.1/heat shock protein 70* (615 aa,

67.09 kDa)

Chaperone, C. neoformans

immunogen (25)

Cap67, 2E-TUC, H99

5 CNAG_02292.1/superoxide dismutase copper

chaperone* (288 aa, 30.32 kDa)

Antioxidant defense, C. neoformans

virulence (9, 34)

Cap67

6 CNAG_02944.1/acid phosphatase (552 aa, 59.32

kDa)

Adhesion of C. neoformans to

epithelial cells (8)

Cap67, 2E-TUC

7 CNAG_02801.1/thioredoxin (105 aa, 11.44 kDa)* Protection against oxidative stress,

survival in the oxidative

environment of macrophages, C.

neoformans virulence (29)

Cap67, H99

8 CNAG_05847.1/thioredoxin reductase (372 aa,

39.84 kDa)

C. neoformans viability (30) Cap67

9 CNAG_06917.1/thiol-speciﬁc antioxidant protein

(234 aa, 25.90 kDa)

C. neoformans virulence, resistance

to nitric oxide and peroxide (31)

H99, 2E-TUC, Cap67

10 CNAG_00575.1/catalase A (683 aa, 76.42 kDa) C. neoformans antioxidant defense

(22)

2E-TUC

11 CNAG_03322.1/UDP-glucuronic acid

decarboxylase Uxs1p (411 aa, 46.47 kDa)

Converts UDP-glucuronic acid to

UDP-xylose, capsule synthesis in

C. neoformans (3)

Cap67

12 CNAG_04969.1/UDP-glucose dehydrogenase

(469 aa, 51.36 kDa)

C. neoformans growth at 37°C and

capsule biosynthesis (33)

Cap67, H99

Signal transduction

13 CNAG_04762.1/Ras2 (364 aa, 39.47 kDa) Mating and high-temp growth in C.

neoformans (52)

Cap67

14 CNAG_03315.1/Rho1* (198 aa, 21.68 kDa) GTP-binding protein, cell wall

integrity in C. neoformans (19)

Cap67

15 CNAG_05235.1/14-3-3 protein* (257 aa, 28.98

kDa)

Cell cycle regulation in C.

neoformans (53)

Cap67, H99, 2E-TUC

16 CNAG_04577.1/nucleoside-diphosphate kinase

(216 aa, 23.36 kDa)

Exchange of phosphate groups

between different nucleoside

diphosphates

Cap67

17 CNAG_03052.1/protein phosphatase type 2C

(560 aa, 59.40 kDa)

Stress response and cell cycle

regulation

Cap67

Ribosomal proteins*

18 CNAG_06447.1/60S ribosomal protein l17 (183

aa, 20.49 kDa)

Ribosomal protein Cap67, H99

19 CNAG_03283.1/60S ribosomal protein L24 (L30)

(151 aa, 16.89 kDa)

Ribosomal protein Cap67

20 CNAG_00232.1/60S ribosomal protein l30-1 (l32)

(114 aa, 12.33 kDa)

Ribosomal protein 2E-TUC

21 CNAG_05762.1/ribosomal protein P2 (112 aa,

11.07 kDa)

Ribosomal protein Cap67

22 CNAG_00771.1/ribosomal protein L35 (128 aa,

14.67 kDa)

Ribosomal protein Cap67

23 CNAG_00779.1/ribosomal protein L27 (137 aa,

15.76 kDa)

Ribosomal protein Cap67

24 CNAG_06605.1/ribosomal protein S2 (257 aa,

27.62 kDa)

Ribosomal protein Cap67

25 CNAG_02144.1/60S ribosomal protein l1-a (l10a)

(225 aa, 25.50 kDa)

Ribosomal protein Cap67, 2E-TUC

26 CNAG_04114.1/40S ribosomal protein S0 (293

aa, 31.46 kDa)

Ribosomal protein Cap67

27 CNAG_01480.1/ribosomal protein L12 (166 aa,

17.55 kDa)

Ribosomal protein Cap67

28 CNAG_06633.1/40S ribosomal protein S15 (151

aa, 17.12 kDa)

Ribosomal protein Cap67

29 CNAG_00656.1/60S ribosomal protein l7 (251

aa, 28.25 kDa)

Ribosomal protein Cap67

Continued on following page

OL. 7, 2008 C. NEOFORMANS VESICLES AND VIRULENCE PROTEINS 61

TABLE 1—Continued

Hit no. Accession no./identiﬁcation Function (reference) Strain(s)

30 CNAG_00034.1/60S ribosomal protein l9 (192

aa, 21.17 kDa)

Ribosomal protein Cap67

31 CNAG_06095.1/ribosomal protein L13 (206 aa,

23.31 kDa)

Ribosomal protein Cap67

32 CNAG_04011.1/60S ribosomal protein l37a (93

aa, 10.07 kDa)

Ribosomal protein Cap67

33 CNAG_02818.1/ribosomal protein S11 (410 aa,

44.31 kDa)

Ribosomal protein Cap67

Sugar and lipid

metabolism

34 CNAG_06699.1/glyceraldehyde-3-phosphate

dehydrogenase* (340 aa, 36.28 kDa)

Glucose metabolism, secreted

enzyme

Cap67, 2E-TUC

35 CNAG_00061.1/citrate synthase* (465 aa, 51.02

kDa)

Energy metabolism Cap67, 2E-TUC

36 CNAG_05653.1/malate synthase* (539 aa, 59.96

kDa)

Glucose metabolism 2E-TUC

37 CNAG_03072.1/phosphopyruvate hydratase

(enolase)* (434 aa, 46.44 kDa)

Cell wall constituent in Candida

(40)

Cap67, H99, 2E-TUC

38 CNAG_04659.1/pyruvate decarboxylase* (624

aa,)

Decarboxylation of pyruvic acid to

acetaldehyde and carbon dioxide

Cap67, H99

39 CNAG_03225.1/malate dehydrogenase* (339 aa,

35.58 kDa)

Conversion of malate into

oxaloacetate, major antigen in P.

brasiliensis (12)

Cap67

40 CNAG_02620.1/phosphogluconate

dehydrogenase* (decarboxylating) (492 aa,

53.85 kDa)

Pentose phosphate pathway Cap67, 2E-TUC

41 CNAG_03920.1/isocitrate dehydrogenase* (453

aa, 50.60 kDa)

Citric acid cycle Cap67

42 CNAG_02100.1/fatty-acid synthase complex

protein* (1,439 aa, 157.47 kDa)

Fatty acid biosynthetic process Cap67

43 CNAG_06140.1/long-chain fatty acid transporter

(111 aa, 11.91 kDa)

Fatty acid biosynthetic process Cap67

44 CNAG_06572.1/pyruvate dehydrogenase e1

component alpha subunit, mitochondrial

precursor (414 aa, 45.66 kDa)

Conversion of pyruvate to acetyl

coenzyme A and CO

Cap67

Nuclear proteins

45 CNAG_01648.1/histone H4* (104 aa, 11.40 kDa) DNA assembly H99, Cap67, 2E-TUC

46 CNAG_05221.1/histone H2A* variant (139 aa,

14.70 kDa)

DNA assembly Cap67, 2E-TUC

Protein/amino acid

metabolism

47 CNAG_00370.1/ubiquitin-carboxy extension

protein fusion* (130 aa, 14.64 kDa)

Posttranslational protein

modiﬁcation

H99, 2E-TUC

48 CNAG_02500.1/endoplasmic reticulum-

associated protein, catabolism-related protein

(552 aa, 60.97 kDa), similar to fungal calnexin

Retention of unfolded or

unassembled N-linked

glycoproteins in the endoplasmic

reticulum

Cap67, H99

49 CNAG_00441.1/inosine 5-monophosphate

dehydrogenase (545 aa, 57.85 kDa)

Oxidation of inosine 5Ј-

monophosphate to xanthosine

5Ј-monophosphate

Cap67

50 CNAG_01890.1/5-

methyltetrahydropteroyltriglutamate-

homocysteine S-methyltransferase (764 aa,

85.32 kDa)

Amino acid metabolism,

cobalamin-independent

methionine synthase

Cap67, H99

51 CNAG_05725.1/acetohydroxy acid

reductoisomerase (402 aa, 44.31 kDa)

Amino acid metabolism Cap67

52 CNAG_00457.1/glutamine synthetase (359 aa,

39.49 kDa)

Amino acid metabolism Cap67

53 CNAG_00785.1/translation initiation factor* (402

aa, 45.13 kDa)

Protein synthesis Cap67

54 CNAG_00930.1/argininosuccinate synthase (430

aa, 47.34 kDa)

Amino acid metabolism Cap67

55 CNAG_04601.1/glycine hydroxymethyltransferase

(500 aa, 54.39 kDa)

Amino acid metabolism Cap67, H99

Continued on following page

62 RODRIGUES ET AL. E

UKARYOT.CELL

areas of serological reaction were also observed in the molec-

ular mass ranges of 50 to 65 and 70 to 100 kDa. These results

suggest that vesicle-related cryptococcal proteins are produced

during human infection and also that potentially effective pro-

tein immunogens are present in extracellular vesicles. Unfor-

tunately, the identiﬁcation of the proteins recognized by the

sera of cryptococcosis patients has been hampered by the lack

of sufﬁcient amount of vesicular material needed for liquid

chromatography-tandem MS sequencing from SDS-PAGE gel

bands.

Proteomic analysis of the C. neoformans extracellular vesi-

cles. To further characterize the protein components of the

secreted vesicles, the material puriﬁed by centrifugation was

enzymatically digested, fractionated by chromatographic meth-

ods, and analyzed by liquid chromatography-tandem MS.

Overall, 76 different proteins were identiﬁed (Table 1). The

TABLE 1—Continued

Hit no. Accession no./identiﬁcation Function (reference) Strain(s)

56 CNAG_00450.1/3-isopropylmalate dehydrogenase

(374 aa, 39.85 kDa)

Amino acid metabolism Cap67

57 CNAG_00992.1/homocitrate synthase (490 aa,

53.51 kDa)

Amino acid metabolism Cap67

58 CNAG_06125.1/translation elongation factor 1␣*

(461 aa, 50.32 kDa)

Protein synthesis Cap67, H99, 2E-TUC

59 CNAG_06908.1/aminomethyltransferase,

mitochondrial precursor (338 aa, 34.92 kDa)

Amino acid metabolism Cap67

Plasma membrane

proteins

60 CNAG_06101.1/ADP/ATP carrier (314 aa, 33.77

kDa)

ATP/ADP translocation, multipass

membrane protein

Cap67, 2E-TUC

61 CNAG_06400.1/plasma membrane H(ϩ)-ATPase

(999 aa, 108.47 kDa)

Proton pump Cap67

62 CNAG_03058.1/Hmp1 protein (115 aa, 11.74

kDa)

Similar to ␣-catenins, proteins

found in complexes with

cadherin cell adhesion

2E-TUC

63 CNAG_04758.1/ammonium transporter (497 aa,

53.04 kDa)

Multipass membrane protein Cap67

Cytoskeleton

proteins

64 CNAG_00483.1/actin* (378 aa, 42.02 kDa) Cytoskeleton protein Cap67, 2E-TUC, H99

65 CNAG_03787.1/␣-tubulin* (449 aa, 49.67 kDa) Cytoskeleton protein Cap67

66 CNAG_01840.1/tubulin ␤ chain* (452 aa, 49.99

kDa)

Cytoskeleton protein Cap67

Miscellaneous

67 CNAG_01577.1/glutamate dehydrogenase (452

aa, 49.16 kDa)

Urea synthesis Cap67, 2E-TUC

68 CNAG_05750.1/ATP synthase ␣ chain,

mitochondrial precursor (541 aa, 58.02 kDa)

ATP synthesis Cap67, H99, 2E-TUC

69 CNAG_05918.1/ATP synthase ␤ chain* (548 aa,

58.63 kDa)

ATP synthesis Cap67, 2E-TUC

70 CNAG_02974.1/voltage-dependent ion-selective

channel (293 aa, 30.61 kDa)

Membrane transport Cap67, 2E-TUC, H99

71 CNAG_01539.1/inositol-3-phosphate synthase

(559 aa, 61.34 kDa)

Metabolism of inositol-containing

molecules

Cap67

72 CNAG_05909.1/electron transporter, transferring

electrons within CoQH2-cytochrome c

reductase complex (320 aa, 34.97 kDa)

Mitochondrial protein, electron

transport

2E-TUC

73 CNAG_00799.1/hypothetical protein similar to

glycosyl hydrolase family 5 protein from

Stigmatella aurantiaca (790 aa, 85.25 kDa)

Cell wall assembly (putative) 2E-TUC

74 CNAG_03537.1/FA and derivative metabolism-

related protein (1022 aa, 108.90 kDa)

FA metabolism Cap67, H99

75 CNAG_00965.1/probable transketolase (688 aa,

74.28 kDa)

NADPH metabolism Cap67, H99

76 CNAG_05759.1/acetyl coenzyme A carboxylase

(2238 aa, 247.95 kDa)

Synthesis of malonyl coenzyme A Cap67

Biological functions of the proteins detected by proteomics are referenced when an association of the molecule with the biology or pathogenesis of C. neoformans

or other fungal pathogens was previously described. General functions in other models were obtained from the ExPASy Proteomics Server (http://ca.expasy.org/). The

number of amino acids (aa) and molecular mass values are presented for each protein. Twenty-ﬁve proteins with unknown functions were characterized but not

presented in this table. For detailed information, see the supplemental material. Proteins that were already characterized in extracellular vesicles produced by

mammalian cells are marked with an asterisk. For details, see references 1, 21, 28, 42, 48, and 49.

VOL. 7, 2008 C. NEOFORMANS VESICLES AND VIRULENCE PROTEINS 63

yield of protein detection varied considerably, depending on

the presence of capsular structures, suggesting that the poly-

saccharides in extracellular fractions might interfere with pro-

tein digestion, as previously suggested for Aspergillus oryzae

(38). Accordingly, no proteins were detected by proteomic

approaches in preliminary analysis of vesicles from the highly

encapsulated strain 24067 (data not shown). Twenty different

proteins were identiﬁed using strain H99, which presents

smaller capsules than isolate 24067. A higher number of pro-

teins (n ϭ 26) was identiﬁed using the encapsulated strain

2E-TUC, but protein characterization was maximum using the

acapsular mutant Cap 67 (70 proteins). The results obtained

with each strain were combined and are summarized in Table

1. A complete, detailed list of proteins analyzed by mass spec-

trometry is available (see Table S1 in the supplemental mate-

rial). Chaperones, including heat shock proteins (Hsp70 and

Hsp90) and superoxide dismutase, signal transduction regula-

tors, antioxidant and cytosolic proteins, and enzymes were

identiﬁed. Of note, nuclear proteins such as histones and ri-

bosomal proteins were also identiﬁed, as previously described

in vesicles secreted by mammalian cells (1, 42). Overall, 27 of

the 76 proteins identiﬁed in the C. neoformans vesicles were

already reported as vesicular proteins in mammalian exosomes

(1, 21, 28, 42, 48, 49), including a ubiquitin-related protein.

Figure 4 shows identiﬁed C. neoformans vesicle proteins dis-

tributed according to their functions in fungal cells.

Intracellular distribution of vesicles in C. neoformans. Mam-

malian exosomes consist of secreted vesicles that originate

from key compartments of the endocytic pathway, namely mul-

tivesicular bodies (MVBs) (51). Since the proﬁle of protein

detection in the C. neoformans vesicles resembled those found

in mammalian exosomes, ultrathin sections of yeast cells were

searched for the presence of MVB-like structures. Vesicular

structures with dimensions similar to those found at the extra-

cellular fractions were observed inside vacuole-like compart-

ments (Fig. 5), resembling endosome-derived MVBs (51).

These vesicle-containing vacuoles were detected in all the sec-

tions analyzed, independently of the technique used for TEM

(data not shown). Bilayered membranes were present at the

vacuole-like structures, which were sometimes found in fusion

with the plasma membrane. These results suggest that vesicle

secretion in C. neoformans may derive from MVBs or vesicle-

containing vacuoles.

DISCUSSION

The secretion of virulence factors is a mechanism used by

different pathogens to cause damage to host cells (6). In C.

neoformans, secreted virulence factors include superoxide dis-

mutase (9), phospholipase B (10), urease (11), and GXM, a

high-molecular-weight polysaccharide (reviewed in references

26 and 32). GXM is synthesized in intracellular compartments

(17, 20, 54) and transported through the cell wall in secretory

vesicles (45). The virulence regulator glucosylceramide (44) is

also present in the cryptococcal extracellular vesicles (45), but

the presence of other molecules in these membrane compart-

ments is unknown. In this context, we hypothesized that vesic-

ular secretion in C. neoformans could be a general secretory

mechanism, possibly used for the delivery of different mole-

cules related to pathogenic mechanisms to the extracellular

space.

Polysaccharide-containing vesicles in C. neoformans ap-

peared to be associated with the Golgi apparatus-derived se-

cretory pathway (54). Golgi apparatus-derived secretory vesi-

cles, however, are expected to fuse with the plasma membrane

and release their internal content to the extracellular space

(41). In C. neoformans, this model would satisfactorily explain

how GXM reaches the periplasmic space, although it does not

explain how the polysaccharide would reach the outer layer of

the cell wall to be incorporated into the growing capsule. Fur-

thermore, such a mechanism could not account for the origin

of the GXM-containing extracellular vesicles that we recently

described. In fact, the existence of GXM-containing extracel-

lular vesicles implies the existence of a vesicle secretion mech-

anism whereby there is no fusion of secretory vesicles and the

plasma membrane. In this context, we examined whether the

secretory vesicles of C. neoformans could have a relationship

with exosomes, which are deﬁned as non-plasma-membrane-

derived vesicles (18).

Exosomes are the only type of bioactive vesicles originating

from an intracellular compartment, named MVBs on the basis

of their morphology (18, 51). These intracellular compart-

ments are derived from endosomes and have well-known func-

tions as intermediates in the degradation of proteins internal-

ized from the cell surface or sorted from the trans-Golgi

network (18, 51). Internal vesicles of MVBs are generated by

budding from the limiting membrane into the lumen of endo-

somes (51). In the degradation pathway, MVBs fuse with ly-

sosomes. However, in several hematopoietic and nonhemato-

poietic cells, MVBs fuse with the plasma membrane, resulting

in the release of internal vesicles to the extracellular milieu as

exosomes (18, 51). In this context, we evaluated whether MVB-

like compartments were present in C. neoformans cells. In fact,

several vacuole-like compartments containing vesicles with di-

mensions similar to those of exosomes and the C. neoformans

extracellular vesicles were observed. Some of these vesicle-

containing vacuoles were in close association with the plasma

membrane and the cell wall, suggesting that the release of

extracellular vesicles to the extracellular space in C. neofor-

mans involves MVB-like compartments. Since endosomes and

MVBs can be connected to the trans-Golgi secretory pathway

(51), we speculate that the previously described Golgi appara-

tus-derived vesicles containing GXM (54) are linked to MVBs

in C. neoformans, which would result in the release of polysac-

FIG. 4. Functional classiﬁcation of the C. neoformans vesicle proteins.

The number of proteins found for each class is shown. Unidentiﬁed

proteins are not shown. For details, see Table S1 in the supplemental

material.

64 RODRIGUES ET AL. E

UKARYOT.CELL

charide-containing vesicles into the periplasmic space. In ac-

cordance with this supposition, treatment of C. neoformans

with brefeldin A, an inhibitor of the Golgi apparatus-derived

transport of molecules, results in a signiﬁcant inhibition of

capsule expression (23).

Fungal proteins frequently have more than a single function

and are found in different cellular locations (2, 4, 16, 37). For

example, histones have been described as being present at the

cell wall of Histoplasma capsulatum, where they are targeted by

antifungal antibodies (37). Glyceraldehyde-3-phosphate dehy-

drogenase, a major protein component of the glycolytic path-

way, is present in the cell wall of Paracoccidioides brasiliensis,

where it participates in the pathogenic processes mediating the

adhesion of yeast cells to host cells and the extracellular matrix

(2). In the same model, the mitochondrial protein Mdj1 was

detected not only in the mitochondria, where it is apparently

sorted, but also in the cell wall (4). Proteomic analysis of the C.

neoformans vesicles revealed a complex protein composition

that included chaperone and membrane, cytoplasmic, and even

nuclear and mitochondrial proteins. Several of these proteins

were similar to those described in mammalian exosomes, which

usually contain cytoplasmic proteins such as elongation factors,

tubulin, actin, actin-binding proteins, annexins, and Rab pro-

tein, molecules responsible for signal transduction, and heat-

shock proteins such as Hsp70 and Hsp90 (1, 21, 28, 42, 48, 49).

Sorting of cytosolic proteins into exosomes is normally ex-

plained by a random engulfment of small portions of cytosol

during the inward budding process of MVBs (51). These ob-

servations, together with morphological data, could support

the supposition that the extracellular vesicles produced by C.

neoformans are exosome-like structures.

Protein composition and morphological analyses indicate

that the C. neoformans extracellular vesicles are not a uniform

population. Vesicles with clearly different electron densities

were observed, and some of them were observed to carry

pigment-like structures. The observation of electron-dense

spots in the inner vesicle compartments suggests the presence

of the molecular machinery necessary for the synthesis of mel-

anin, a pigment that has been concretely associated with the

virulence of C. neoformans (46). Melanin is autopolymerized

from the oxidation of diphenolic compounds by the enzyme

laccase (36). In this context, we incubated the vesicular sus-

pension with the laccase substrate

L-DOPA, which demon-

strated laccase activity in C. neoformans vesicles. The ﬁnding of

FIG. 5. TEM of C. neoformans suggesting the presence of cytoplasmic vacuole-containing vesicles reminiscent of exosome-like structures. (A) Over-

view of a C. neoformans cell with different cytoplasmic vacuoles containing vesicles (black asterisks). The white asterisk indicates the cell wall. Scale bar,

500 nm. A magniﬁed view of the vesicle-containing vacuoles is shown. Panel B demonstrates that these structures are surrounded by a bilayered

membrane, which sometimes invaginates (arrow). A close association with the cell wall (white asterisk) was observed, suggesting fusion with the plasma

membrane. Scale bar, 200 nm. (C) Intracellular and extracellular vesicles (black arrows) have similar dimensions. Scale bar, 200 nm.

OL. 7, 2008 C. NEOFORMANS VESICLES AND VIRULENCE PROTEINS 65

laccase in vesicles that are possibly derived from MVB has an

intriguing parallel in mammalian systems where tyrosinase-

containing melanosomes are shed from melanocytes after syn-

thesis from early endosomal vesicles (43). However, laccase

was not detected by the proteomic approach. This observation

is probably a false-negative result related to a low protein

concentration, since pigmented vesicles are the less-abundant

fraction in the vesicle population (15%). A relatively low pro-

tein concentration could also explain why urease is also detect-

able by a sensitive enzymatic colorimetric assays but not by

proteomic approaches. Hence, we postulate that the proteins

identiﬁed here may be only a subset of the total proteins found

in vesicles.

We identiﬁed several virulence-related molecules in C. neo-

formans vesicles. This group of molecules includes well-known

virulence factors such as GXM and glucosylceramide, which

were characterized as vesicle components in a previous study

(45). In the present study, we demonstrate the presence of

several other components associated with virulence in vesicular

fractions, such as enzymes related to capsule synthesis (3),

urease (11), laccase (46), acid phosphatase (8), heat shock

proteins (25), and several antioxidant proteins such as super-

oxide dismutase (9, 34), thioredoxin (29), thioredoxin reduc-

tase (30), thiol-speciﬁc antioxidant protein (31), and catalase A

(22). Some of the vesicle proteins were recognized by sera from

cryptococcosis patients, suggesting that these proteins are pro-

duced during human infection. The combined presence of lip-

ids, pigments, polysaccharides, and virulence-related and im-

munogenic proteins suggests that C. neoformans uses vesicular

secretion as a single mechanism to deliver virulence factors

into the fungal extracellular space. Since vesicle production has

been observed in vivo and during macrophage infection (45),

we suggest that the C. neoformans extracellular vesicles func-

tion as “virulence factor delivery bags” that could expressively

inﬂuence the interaction of fungal cells with the host. Different

types of vesicles may carry different types of toxic payloads.

Clearly, the presence of numerous virulence-associated com-

ponents in vesicular preparations would allow C. neoformans to

deliver a toxic concentrated payload to target cells such as

predatory amoebae and macrophages. Vesicle secretion could

also presumably occur in phagosomal spaces and allow delivery

of toxic payloads to cells that have ingested cryptococcal cells.

Vesicular delivery of concentrated virulence-associated com-

ponents could be signiﬁcantly more effective in damaging toxic

cells than if such components were secreted separately and had

to reach target cells through diffusion. In this context, it has

been recently suggested by our group that vesicle secretion

occurs during infection of host macrophages (45). This obser-

vation could be related to the known ability of C. neoformans

to secrete GXM during intracellular infection of phagocytes

(50), which results in host cell toxicity and release of fungal

cells to extracellular host sites.

In summary, we report the identiﬁcation of numerous

virulence-associated components in C. neoformans vesicle

preparations. The similarities in the protein content of C.

neoformans vesicles and mammalian exosome-like struc-

tures combined with electron microscopic morphological evi-

dence of exosome-like structures in cryptococcal cells led us to

propose that the extracellular vesicles originate from fungal

exosomes. The complexity of vesicle populations with respect

to their morphology and cargo suggest numerous new avenues

for the investigation of their role in virulence and cryptococcal

cell biology.

ACKNOWLEDGMENTS

M.L.R. and L.N. are supported by grants from the Brazilian agencies

CNPq, CAPES, FAPESP, and FAPERJ. A.C. is supported by NIH

grants AI033142, AI033774, AI052733, and HL059842. I.C.A. is sup-

ported by NIH grant 5G12RR008124 (to the Border Biomedical Re-

search Center [BBRC]/University of Texas at El Paso [UTEP]). J.D.N.

is supported by NIH AI056070-01A2 and AI52733. We are thankful to

the Biomolecule Analysis Core Facility/BBRC/UTEP, supported by

NIH/NCRR grant 5G12RR008124. D.L.O. is a Ph.D. student at the

Instituto de Bioquimica Medica, UFRJ.

We thank Kildare Miranda and the Albert Einstein College of

Medicine Analytical Imaging Facility staff for help with the electron

microscopy. We are also indebted to Fabio Gozzo (Laboratorio Na-

cional de Luz Sincrontron, Campinas, Brazil) for the 100,000 random

sequences used for statistics in the proteomic analysis.

REFERENCES

1. Aoki, N., S. Jin-no, Y. Nakagawa, N. Asai, E. Arakawa, N. Tamura, T.

Tamura, and T. Matsuda. 2007. Identiﬁcation and characterization of mi-

crovesicles secreted by 3T3-L1 adipocytes: redox- and hormone-dependent

induction of milk fat globule-epidermal growth factor 8-associated mi-

crovesicles. Endocrinology 148:3850–3862.

2. Barbosa, M. S., S. N. Bao´, P. F. Andreotti, F. P. de Faria, M. S. S. Felipe, L.

dos Santos Feitosa, M. J. S. Mendes-Giannini, and C. M. d. A. Soares. 2006.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis

is a cell surface protein involved in fungal adhesion to extracellular matrix

proteins and interaction with cells. Infect. Immun. 74:382–389.

3. Bar-Peled, M., C. L. Grifﬁth, and T. L. Doering. 2001. Functional cloning

and characterization of a UDP-glucuronic acid decarboxylase: the patho-

genic fungus Cryptococcus neoformans elucidates UDP-xylose synthesis.

Proc. Natl. Acad. Sci. USA 98:12003–12008.

4. Batista, W. L., A. L. Matsuo, L. Ganiko, T. F. Barros, T. R. Veiga, E.

Freymu¨ller, and R. Puccia. 2006. The PbMDJ1 gene belongs to a conserved

MDJ1/LON locus in thermodimorphic pathogenic fungi and encodes a heat

shock protein that localizes to both the mitochondria and cell wall of Para-

coccidioides brasiliensis. Eukaryot. Cell 5:379–390.

5. Bicanic, T., and T. S. Harrison. 2004. Cryptococcal meningitis. Br. Med.

Bull. 72:99–118.

6. Casadevall, A., and L. A. Pirofski. 2003. The damage-response framework of

microbial pathogenesis. Nat. Rev. Microbiol. 1:17–24.

7. Casadevall, A., W. Cleare, M. Feldmesser, A. Glatman-Freedman, D. L.

Goldman, T. R. Kozel, N. Lendvai, J. Mukherjee, L.-A. Pirofski, J. Rivera,

A. L. Rosas, M. D. Scharff, P. Valadon, K. Westin, and Z. Zhong. 1998.

Characterization of a murine monoclonal antibody to Cryptococcus neofor-

mans polysaccharide that is a candidate for human therapeutic studies.

Antimicrob. Agents Chemother. 42:1437–1446.

8. Collopy-Junior, I., F. F. Esteves, L. Nimrichter, M. L. Rodrigues, C. S.

Alviano, and J. R. Meyer-Fernandes. 2006. An ectophosphatase activity in

Cryptococcus neoformans. FEMS Yeast Res. 6:1010–1017.

9. Cox, G. M., T. S. Harrison, H. C. McDade, C. P. Taborda, G. Heinrich, A.

Casadevall, and J. R. Perfect. 2003. Superoxide dismutase inﬂuences the

virulence of Cryptococcus neoformans by affecting growth within macro-

phages. Infect. Immun. 71:173–180.

10. Cox, G. M., H. C. McDade, S. C. Chen, S. C. Tucker, M. Gottfredsson, L. C.

Wright, T. C. Sorrell, S. D. Leidich, A. Casadevall, M. A. Ghannoum, and

J. R. Perfect. 2001. Extracellular phospholipase activity is a virulence factor

for Cryptococcus neoformans. Mol. Microbiol. 39:166–175.

11. Cox, G. M., J. Mukherjee, G. T. Cole, A. Casadevall, and J. R. Perfect. 2000.

Urease as a virulence factor in experimental cryptococcosis. Infect. Immun.

68:443–448.

12. Da Fonseca, C. A., R. S. Jesuino, M. S. Felipe, D. A. Cunha, W. A. Brito, and

C. M. Soares. 2001. Two-dimensional electrophoresis and characterization

of antigens from Paracoccidioides brasiliensis. Microbes Infect. 3:535–542.

13. Datta, K., and L. A. Pirofski. 2006. Towards a vaccine for Cryptococcus

neoformans: principles and caveats. FEMS Yeast Res. 6:525–536.

14. De Souza Pereira, R., and J. Geibel. 1999. Direct observation of oxidative

stress on the cell wall of Saccharomyces cerevisiae strains with atomic force

microscopy. Mol. Cell. Biochem. 201:17–24.

15. Eng, J. K., A. L. McCormack, and J. R. Yates III. 1994. An approach to

correlate tandem mass spectral data of peptides with amino acid sequences

in a protein database. J. Am. Soc. Mass Spectrom. 5:976–989.

16. Esnault, K., B. el Moudni, J.-P. Bouchara, D. Chabasse, and G. Tronchin.

1999. Association of a myosin immunoanalogue with cell envelopes of As-

66 RODRIGUES ET AL. EUKARYOT.CELL

pergillus fumigatus conidia and its participation in swelling and germination.

Infect. Immun. 67:1238–1244.

17. Feldmesser, M., Y. Kress, and A. Casadevall. 2001. Dynamic changes in the

morphology of Cryptococcus neoformans during murine pulmonary infection.

Microbiology 147:2355–2365.

18. Fevrier, B., and G. Raposo. 2004. Exosomes: endosomal-derived vesicles

shipping extracellular messages. Curr. Opin. Cell Biol. 16:415–421.

19. Fuchs, B. B., G. P. Tegos, M. R. Hamblin, and E. Mylonakis. 2007. Suscep-

tibility of Cryptococcus neoformans to photodynamic inactivation is associ-

ated with cell wall integrity. Antimicrob. Agents Chemother. 51:2929–2936.

20. Garcia-Rivera, J., Y. C. Chang, K. J. Kwon-Chung, and A. Casadevall. 2004.

Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular

trafﬁcking of capsular glucuronoxylomannan. Eukaryot. Cell 3:385–392.

21. Gatti, J. L., S. Metayer, M. Belghazi, F. Dacheux, and J. L. Dacheux. 2005.

Identiﬁcation, proteomic proﬁling, and origin of ram epididymal ﬂuid exo-

some-like vesicles. Biol. Reprod. 72:1452–1465.

22. Giles, S. S., J. E. Stajich, C. Nichols, Q. D. Gerrald, J. A. Alspaugh, F.

Dietrich, and J. R. Perfect. 2006. The Cryptococcus neoformans catalase gene

family and its role in antioxidant defense. Eukaryot. Cell 5:1447–1459.

23. Hu, G., B. R. Steen, T. Lian, A. P. Sham, N. Tam, K. L. Tangen, and J. W.

Kronstad. 2007. Transcriptional regulation by protein kinase A in Crypto-

coccus neoformans. PLoS Pathog. 3:e42.

24. Jurado, J. D., E. D. Rael, C. S. Lieb, E. Nakayasu, W. K. Hayes, S. P. Bush,

and J. A. Ross. 2007. Complement inactivating proteins and intraspecies

venom variation in Crotalus oreganus helleri. Toxicon 49:339–350.

25. Kakeya, H., H. Udono, S. Maesaki, E. Sasaki, S. Kawamura, M. A. Hossain,

Y. Yamamoto, T. Sawai, M. Fukuda, K. Mitsutake, Y. Miyazaki, K. Tomono,

T. Tashiro, E. Nakayama, and S. Kohno. 1999. Heat shock protein 70

(hsp70) as a major target of the antibody response in patients with pulmo-

nary cryptococcosis. Clin. Exp. Immunol. 115:485–490.

26. McFadden, D., O. Zaragoza, and A. Casadevall. 2006. The capsular dynam-

ics of Cryptococcus neoformans. Trends Microbiol. 14:497–505.

27. McFadden, D. C., M. De Jesus, and A. Casadevall. 2006. The physical

properties of the capsular polysaccharides from Cryptococcus neoformans

suggest features for capsule construction. J. Biol. Chem. 281:1868–1875.

28. Mears, R., R. A. Craven, S. Hanrahan, N. Totty, C. Upton, S. L. Young, P.

Patel, P. J. Selby, and R. E. Banks. 2004. Proteomic analysis of melanoma-

derived exosomes by two-dimensional polyacrylamide gel electrophoresis

and mass spectrometry. Proteomics 4:4019–4031.

29. Missall, T. A., and J. K. Lodge. 2005. Function of the thioredoxin proteins in

Cryptococcus neoformans during stress or virulence and regulation by puta-

tive transcriptional modulators. Mol. Microbiol. 57:847–858.

30. Missall, T. A., and J. K. Lodge. 2005. Thioredoxin reductase is essential for

viability in the fungal pathogen Cryptococcus neoformans. Eukaryot. Cell

4:487–489.

31. Missall, T. A., J. M. Moran, J. A. Corbett, and J. K. Lodge. 2005. Distinct

stress responses of two functional laccases in Cryptococcus neoformans are

revealed in the absence of the thiol-speciﬁc antioxidant Tsa1. Eukaryot. Cell

4:202–208.

32. Monari, C., F. Bistoni, and A. Vecchiarelli. 2006. Glucuronoxylomannan

exhibits potent immunosuppressive properties. FEMS Yeast Res. 6:537–542.

33. Moyrand, F., and G. Janbon. 2004. UGD1, encoding the Cryptococcus neo-

formans UDP-glucose dehydrogenase, is essential for growth at 37°C and for

capsule biosynthesis. Eukaryot. Cell 3:1601–1608.

34. Narasipura, S. D., J. G. Ault, M. J. Behr, V. Chaturvedi, and S. Chaturvedi.

2003. Characterization of Cu, Zn superoxide dismutase (SOD1) gene knock-

out mutant of Cryptococcus neoformans var. gattii: role in biology and viru-

lence. Mol. Microbiol. 47:1681–1694.

35. Nimrichter, L., M. L. Rodrigues, E. G. Rodrigues, and L. R. Travassos. 2005.

The multitude of targets for the immune system and drug therapy in the

fungal cell wall. Microbes Infect. 7:789–798.

36. Nosanchuk, J. D., and A. Casadevall. 2006. Impact of melanin on microbial

virulence and clinical resistance to antimicrobial compounds. Antimicrob.

Agents Chemother. 50:3519–3528.

37. Nosanchuk, J. D., J. N. Steenbergen, L. Shi, G. S. Deepe, Jr., and A.

Casadevall. 2003. Antibodies to a cell surface histone-like protein protect

against Histoplasma capsulatum. J. Clin. Investig. 112:1164–1175.

38. Oda, K., D. Kakizono, O. Yamada, H. Iefuji, O. Akita, and K. Iwashita. 2006.

Proteomic analysis of extracellular proteins from Aspergillus oryzae grown

under submerged and solid-state culture conditions. Appl. Environ. Micro-

biol. 72:3448–3457.

39. Perfect, J. R., and A. Casadevall. 2002. Cryptococcosis. Infect. Dis. Clin. N.

Am. 16:837–874, v–vi.

40. Pitarch, A., A. Jimenez, C. Nombela, and C. Gil. 2006. Decoding serological

response to Candida cell wall immunome into novel diagnostic, prognostic,

and therapeutic candidates for systemic candidiasis by proteomic and bioin-

formatic analyses. Mol. Cell Proteomics 5:79–96.

41. Ponnambalam, S., and S. A. Baldwin. 2003. Constitutive protein secretion

from the trans-Golgi network to the plasma membrane. Mol. Membr. Biol.

20:129–139.

42. Potolicchio, I., G. J. Carven, X. Xu, C. Stipp, R. J. Riese, L. J. Stern, and L.

Santambrogio. 2005. Proteomic analysis of microglia-derived exosomes:

metabolic role of the aminopeptidase CD13 in neuropeptide catabolism.

J. Immunol. 175:2237–2243.

43. Raposo, G., and M. S. Marks. 2007. Melanosomes—dark organelles en-

lighten endosomal membrane transport. Nat. Rev. Mol. Cell Biol. 8:786–797.

44. Rittershaus, P. C., T. B. Kechichian, J. C. Allegood, A. H. Merrill, Jr., M.

Hennig, C. Luberto, and M. Del Poeta. 2006. Glucosylceramide synthase is

an essential regulator of pathogenicity of Cryptococcus neoformans. J. Clin.

Investig. 116:1651–1659.

45. Rodrigues, M. L., L. Nimrichter, D. O. Oliveira, S. Frases, K. Miranda, O.

Zaragoza, M. Alvarez, A. Nakouzi, M. Feldmesser, and A. Casadevall. 2007.

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic

solution to the problem of fungal trans-cell wall transport. Eukaryot. Cell

6:48–59.

46. Salas, S. D., J. E. Bennett, K. J. Kwon-Chung, J. R. Perfect, and P. R.

Williamson. 1996. Effect of the laccase gene CNLAC1, on virulence of

Cryptococcus neoformans. J. Exp. Med. 184:377–386.

47. Schmid, F., F. Separovic, B. M. McDougall, B. A. Stone, R. T. Brownlee, and

R. J. Seviour. 2007. Characterisation of the extracellular polysaccharides

produced by isolates of the fungus Acremonium. Carbohydr. Res. [Epub

ahead of print.]

48. Segura, E., S. Amigorena, and C. Thery. 2005. Mature dendritic cells secrete

exosomes with strong ability to induce antigen-speciﬁc effector immune

responses. Blood Cells Mol. Dis. 35:89–93.

49. Thery, C., M. Boussac, P. Veron, P. Ricciardi-Castagnoli, G. Raposo, J.

Garin, and S. Amigorena. 2001. Proteomic analysis of dendritic cell-derived

exosomes: a secreted subcellular compartment distinct from apoptotic vesi-

cles. J. Immunol. 166:7309–7318.

50. Tucker, S. C., and A. Casadevall. 2002. Replication of Cryptococcus neofor-

mans in macrophages is accompanied by phagosomal permeabilization and

accumulation of vesicles containing polysaccharide in the cytoplasm. Proc.

Natl. Acad. Sci. USA 99:3165–3170.

51. van Niel, G., I. Porto-Carreiro, S. Simoes, and G. Raposo. 2006. Exosomes:

a common pathway for a specialized function. J. Biochem. (Tokyo) 140:

13–21.

52. Waugh, M. S., C. B. Nichols, C. M. DeCesare, G. M. Cox, J. Heitman, and

J. A. Alspaugh. 2002. Ras1 and Ras2 contribute shared and unique roles in

physiology and virulence of Cryptococcus neoformans. Microbiology 148:191–

201.

53. Woyke, T., M. E. Berens, D. B. Hoelzinger, G. R. Pettit, G. Winkelmann, and

R. K. Pettit. 2004. Differential gene expression in auristatin PHE-treated

Cryptococcus neoformans. Antimicrob. Agents Chemother. 48:561–567.

54. Yoneda, A., and T. L. Doering. 2006. A eukaryotic capsular polysaccharide is

synthesized intracellularly and secreted via exocytosis. Mol. Biol. Cell 17:

5131–5140.

55. Zaragoza, O., A. Telzak, R. A. Bryan, E. Dadachova, and A. Casadevall.

2006. The polysaccharide capsule of the pathogenic fungus Cryptococcus

neoformans enlarges by distal growth and is rearranged during budding. Mol.

Microbiol. 59:67–83.

VOL. 7, 2008 C. NEOFORMANS VESICLES AND VIRULENCE PROTEINS 67

REVIEW – SPECIAL ISSUE

Correspondence: Marcio L. Rodrigues, Universidade Federal do Rio de Janeiro, Instituto de Microbiologia

Professor Paulo de Góes. Avenida Carlos Chagas Filho, 373, Cidade Universitária CCS, Bloco I. Rio de

Janeiro—RJ, 21941-902, Brazil. Tel: 55 21 25626740; Fax: 55 21 25608344. Email:[email protected]

Copyright in this article, its metadata, and any supplementary data is held by its author or authors. It is published under the

Creative Commons Attribution By licence. For further information go to: http://creativecommons.org/licenses/by/3.0/.

Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism

for the Delivery of Virulence-Associated Macromolecules?

Marcio L. Rodrigues

, Leonardo Nimrichter

, Debora L. Oliveira

, Joshua D.

Nosanchuk

2,3

and Arturo Casadevall

2,3

Laboratório de Estudos Integrados em Bioquímica Microbiana, Instituto de Microbiologia

Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941590, Brazil.

Department

of Microbiology and Immunology and the

Division of Infectious Diseases of the Department

of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY, U.S.A. 10461.

Abstract: Fungal cells are encaged in rigid, complex cell walls. Until recently, there was remarkably little information

regarding the trans-fungal cell wall transfer of intracellular macromolecules to the extracellular space. Recently, several

studies have begun to elucidate the mechanisms that fungal cells utilize to secrete a wide variety of macromolecules through

the cell wall. The combined use of transmission electron microscopy, serology, biochemistry, proteomics and lipidomics

have revealed that the fungal pathogens Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida

parapsilosis and Sporothrix schenckii, as well as the model yeast Saccharomyces cerevisiae, each produces extracellular

vesicles that carry lipids, proteins, polysaccharides and pigment-like structures of unquestionable biological signiﬁ cance.

Compositional analysis of the C. neoformans and H. capsulatum extracellular vesicles suggests that they may function as

‘virulence bags’, with the potential to modulate the host-pathogen interaction in favor of the fungus. The cellular origin of

the extracellular vesicles remains unknown, but morphological and biochemical features indicate that they are similar to

the well-described mammalian exosomes.

Keywords: trans-cell wall transport, extracellular vesicles, secretion

Insights into Secretory Mechanisms and Vesicular Transport Systems

Pathogenic mechanisms and secretory processes in microbes are closely associated. Secreted virulence

factors and potent extracellular immunogens have been described for a number of prokaryotic and

eukaryotic pathogens (Engel et al. 1998; Ghannoum, 1998; Hoegl et al, 1996; Hube, 1996; Liu and

Nizet, 2004; McFadden et al. 2006a; Mitchell, 2006; Rodrigues et al. 2008; Rodrigues et al. 2007).

Therefore, the understanding of secretory mechanisms and their regulation in microbial pathogens may

represent a promising strategy for the design of new drugs and prophylactic agents.

There are numerous secretory pathways in eukaryotic and prokaryotic organisms that utilize diverse

mechanisms for secretion (Gorelick and Shugrue, 2001; Lee et al. 2004; Mazar and Cotter, 2007; Rigel

and Braunstein, 2008; Salama and Schekman, 1995; Wickner and Schekman, 2005). In eukaryotes, the

most well-studied pathway of protein secretion involves vesicular migration from the endoplasmic

reticulum to the trans face of the Golgi and then loading into a complex network of vesicles, the trans-

Golgi reticulum (Glick and Malhotra, 1998). These proteins are sorted in the trans-Golgi network into

transport vesicles that immediately move to and fuse with the plasma membrane, releasing their contents

by exocytosis (Glick and Malhotra, 1998). In this regard, the generation of a collection of temperature-

sensitive mutant Saccharomyces cerevisiae yeast strains (sec mutants in which secretion and cell surface

assembly of proteins were blocked at different steps of the secretory pathway) was extremely important

for the elucidation of the sequential events required for secretion (Novick et al. 1980; Novick and

Schekman, 1979; Schekman, 2002; Schekman et al. 1983; Schekman and Novick, 2004). In these cells,

inhibition of protein secretion at high (non-permissive) temperature is usually accompanied by morpho-

logical and biochemical changes, as well as intracellular vesicle accumulation. Other so-called ‘conven-

tional’ mechanisms of secretion involve, for instance, ATP binding cassette type transporters, which are

common to both eukaryotes and prokaryotes (Davidson and Maloney, 2007; Niimi et al. 2005).

Lipid Insights 2008:2 27–40

Rodrigues et al

Lipid Insights 2008:2

Proteins that do not use the classical ER-Golgi

pathway or membrane transporters can be secreted

through various nonclassical pathways, as

recently reviewed by (Nickel and Seedorf, 2008).

Non-classical protein secretion may require vesicle

release to the extracellular space, in a process that

involves the formation of the so-called exosomes.

During exosome biogenesis, small vesicles are

formed by membrane invagination within endo-

cytic compartments (endosomes). The formation

of internal vesicles in the lumen of endosomes

generates the so-called multivesicular bodies,

which usually fuse with lysosomes in degradation

pathways. However, multivesicular bodies can also

fuse with the plasma membrane, resulting in the

release of internal vesicles to the extracellular

milieu as exosomes (Keller et al. 2006).

In contrast to most eukaryotic cells, fungi and

bacteria are cell wall-containing organisms, making

secretion topologically more complex. The presence

of the cell wall, at the very least, implies the exis-

tence of trans-cell wall mechanisms for the release

of molecules to the extracellular space. In prokary-

otes, the mechanisms of transport of proteins across

the cell wall are multiple. A general protein secre-

tion pathway involving multiple genes (SECA,

SECY, SECE, and SECG) and a number of accessory

proteins has been ﬁ rmly documented (Mori and Ito,

2001; Veenendaal et al. 2004). In Gram-negative

bacteria, secretion systems have been widely

reported (reviewed in (Saier, 2006)). This secretion

machinery operates to release proteins extracellu-

larly and, in the case of some pathogens, to inject

toxins within host cells. At least six types of bacte-

rial secretion systems have been deﬁ ned, i.e. type I

to type VI. Comprehensive reviews on this topic

are available in the literature (Bingle et al. 2008;

Cianciotto, 2005; Craig and Li, 2008; Henderson

et al. 2004). Other protein secretion pathways in

bacteria include two-partner secretion systems

(Mazar and Cotter, 2007), which export large

exoproteins across the outer membranes using

channel-forming β-barrel proteins, twin-arginine

transporters (Sargent, 2007), a membrane-bound

transport apparatus that translocate proteins in a

fully folded conformation, and secretion of extracel-

lular vesicles (Mashburn-Warren et al. 2008).

Vesicle-mediated toxin delivery is a potent virulence

mechanism exhibited by diverse Gram-negative

pathogens.

Trans-cell wall secretion in fungi has remained

poorly explored for many years. Although it is well

known that fungal cells secrete molecules of

different chemical natures and molecular masses,

the mechanisms by which extracellular structures

leave intracellular compartments and cross the cell

wall are virtually unknown. As typical eukaryotic

organisms, fungal cells use conventional pathways

of secretion involving post-Golgi vesicles that fuse

with the plasma membrane to release their cargo

(Schekman, 2002; Schekman and Novick, 2004).

In fact, it is well known that yeast cells continu-

ously secrete a number of enzymes that remain

localized in the periplasm (Wickner and Schekman,

2005). The discovery that the capsular polysac-

charide of C. neoformans had a molecular weight

that could exceed 1 million Daltons (McFadden

et al. 2006b), however, revealed the need for con-

sidering new mechanisms of trans-cell wall trans-

port mechanism that could deliver macromolecules

from the periplasmic space outside of the cell.

Recent studies reported the characterization

of extracellular vesicles in pathogenic and

non-pathogenic species of fungi. C. neoformans,

Histoplasma capsulatum, Candida albicans,

Candida parapsilosis, Sporothrix schenckii and

S. cerevisiae were demonstrated to produce extra-

cellular vesicles containing lipid, polysaccharide

and protein components (Albuquerque et al. 2008;

Rodrigues et al. 2008; Rodrigues et al. 2007).

Therefore, extracellular vesicle secretion may

represent a eukaryotic solution to the problem of

trans-cell wall transport. Remarkably, the vesicles

produced by C. neoformans and H. capsulatum

contain key virulence determinants (Albuquerque

et al. 2008; Rodrigues et al. 2008; Rodrigues et al.

2007), suggesting that, as described for bacteria

(Mashburn-Warren et al. 2008), extracellular

vesicles in fungi may represent an efﬁ cient mech-

anism of virulence factor delivery that may be

crucial for the success of the infection. In this

review, we discuss different models of extracel-

lular vesicle secretion, as well as putative pathways

of biogenesis and the impact of vesicle excretion

on fungal pathogenesis.

Extracellular Vesicles and Trans-Cell

Wall Transport: The Cryptococcus

Neoformans Model of Polysaccharide

and Protein Export

The most distinctive characteristic of the yeast

pathogen C. neoformans is the expression of a

polysaccharide capsule, a common feature of

Trans-cell wall secretion in fungi

Lipid Insights 2008:2

prokaryotic pathogens which is usually not

observed in eukaryotic microbes. Another particu-

larity of C. neoformans is the fact that the synthe-

sis of capsular polysaccharides occurs in the

cytoplasm (Feldmesser et al. 2001; Garcia-Rivera

et al. 2004; Yoneda and Doering, 2006). In

prokaryotes, capsule synthesis usually occurs at

surface and extracellular sites. In Escherichia coli,

for instance, capsular polysaccharides are initially

assembled by enzymes associated with the

plasma membrane (Whitﬁ eld, 2006; Whitﬁ eld

et al. 2003).

The capsule of C. neoformans is primarily

composed of two polysaccharides, namely gluc-

uronoxylomannan (GXM) and galactoxylomannan

(GalXM) (McFadden et al. 2006a). GXM, the best

studied capsular component of C. neoformans, is

the main virulence factor in this pathogen

(McClelland et al. 2005). GXM is not only surface

associated, but also continuously secreted to the

extracellular space where it accumulates in tissues

(McFadden et al. 2006a). Interestingly, GXM has

a molecular mass in the range of 1 to 7 million

Daltons (McFadden et al. 2006b), suggesting the

existence of mechanisms of transport of the poly-

saccharide from intracellular sites to the extracel-

lular environment. Early and recent studies

suggested that the transfer of intracellular polysac-

charides to the extracellular space in fungi could

require vesicular transport.

Vesicular transport in fungi was hinted at in

several earlier reports. Heath and colleagues

showed almost 40 years ago that fungal vesicles

contained cell wall precursors, suggesting that

intracellular vesicles can migrate to the cell surface

to release substrates for wall synthesis (Heath et al.

1971). More recently, the cell wall of C. neoformans

was described as the major cellular site of the

glycosphingolipid glucosylceramide (Rodrigues

et al. 2000), which is a membrane component of

vesicles that migrate from the plasma membrane

to the cell wall (Nimrichter et al. 2005b; Rodrigues

et al. 2007; Rodrigues et al. 2000). Early studies

by Takeo and co-workers also demonstrated that

C. neoformans secrete vesicles outside the cell

membrane (Takeo et al. 1973a, b). Interestingly,

despite the fact that no molecular evidence was

provided at that time, the authors proposed that

“capsular material is synthesized in and released

via the vesicles”. There the story rested for almost

20 years, until Feldmesser et al. used immunogold

electron microscopy and noticed clumped labeling

of the cell wall consistent with vesicular transport

of GXM (Feldmesser et al. 2001). Those observa-

tions were further extended by that of Garcia-

Rivera and colleagues who showed that antibodies

to GXM recognized cytoplasmic and cell wall

clusters resembling vesicles (Garcia-Rivera et al.

2004). In essence, these studies provide support

for the suggestion that the capsular polysaccharide

was synthesized in the cytoplasm and exported to

the exterior of the cell in secretory vesicles that

traversed the cell wall.

Based on the classical studies of Schekman and

Novick on yeast mutants defective in protein

synthesis (Novick et al. 1980; Novick and

Schekman, 1979; Schekman, 2002; Schekman

et al. 1983; Schekman and Novick, 2004), Yoneda

and Doering (Yoneda and Doering, 2006) used site-

directed mutagenesis to generate a C. neoformans

strain defective in the production of Sav1p, a

homolog of the S. cerevisiae small GTPase Sec4p.

In S. cerevisiae, sec4 mutants accumulate post-

Golgi vesicles under restrictive conditions

(Walworth et al. 1989), a morphological feature

that was also observed in the C. neoformans mutant

(Yoneda and Doering, 2006). More precisely, the

sav1 mutant of C. neoformans had defective

protein secretion and accumulated exocytic vesi-

cles at the septum and the bud during cell division.

Strikingly, these vesicles were strongly recognized

by an antibody to GXM (Yoneda and Doering,

2006), confirming and extending previous

suppositions that the polysaccharide was synthe-

sized intracellularly and secreted in vesicles.

Since the capsule of C. neoformans enlarges by

apical growth (Zaragoza et al. 2006), one might

deduce that capsular components are secreted to

the extracellular space and incorporated into the

external layers of the growing capsule. Therefore,

assuming that secretion of intracellularly synthe-

sized GXM is an essential element of capsular

growth, one might hypothesize that extracellular

vesicles ﬁ lled up with capsular components may

exist in C. neoformans culture supernatants.

Indeed, cell wall-bound vesicles, as well as extra-

cellular vesicles in association with surface struc-

tures, were observed in vitro and during animal

infection by C. neoformans (Rodrigues et al. 2007).

In addition, the fractionation of culture superna-

tants using centrifugation procedures previously

designed for the puriﬁ cation of secreted vesicles

in mammalian systems allowed the isolation of

extracellular lipid-containing fractions. The analysis

Rodrigues et al

Lipid Insights 2008:2

of these fractions by transmission electron

microscopy revealed the presence of round vesi-

cles, in the range of 20 to 400 nm, containing

bilayered membranes (Rodrigues et al. 2008;

Rodrigues et al. 2007). Vesicle morphology

included usually round compartments with differ-

ent levels of electron-density. Vesicular structures

with hyper-dense structures resembling a dark

pigment were also observed. Importantly, some of

the isolated vesicles were recognized by an anti-

body to GXM (Rodrigues et al. 2007), conﬁ rming

that these compartments were involved in polysac-

charide trans-cell wall secretion. In addition, GXM

detection in vesicles was correlated with capsule

enlargement, suggesting that polysaccharide deliv-

ery in extracellular vesicles is necessary for assem-

bly of the capsule (Rodrigues et al. 2007).

The production of extracellular vesicles requires

cell viability (Rodrigues et al. 2007), conﬁ rming

that these compartments are actively secreted

rather than passively released by dead cells. Trans-

mission electron microscopy of intact C. neofor-

mans cells also revealed that the secretory vesicles

were recognized by an antibody to glucosylce-

ramide (Rodrigues et al. 2007). Since glucosylce-

ramide is an important regulator of growth and

virulence in C. neoformans (Rittershaus et al. 2006;

Rodrigues et al. 2000), the possibility that the

extracellular vesicles carry bioactive lipids was

raised. The analysis of lipid components of isolated

extracellular vesicles by chromatographic and

spectrometric methods revealed the presence of

glucosylceramide (GlcCer) and other structures

(Rodrigues et al. 2007). By thin-layer chromatog-

raphy, bands with migration rates corresponding

to GlcCer and ergosterol were detected in lipid

extracts of vesicles produced by both acapsular

and encapsulated C. neoformans cells. Vesicle

lipids from encapsulated cells were also examined

by electrospray ionization mass spectrome-

try, revealing a complex lipid composition.

GlcCer analysis in vesicle fractions revealed the

presence of N-2’-hydroxyhexadecanoyl- and N-2’-

hydroxyoctadecanoyl-1-beta-

D-glucopyranosyl-9-

methyl-4,8-sphingadienine. Analysis of sterols

showed that ergosterol and 4,14-dimethylergosta-

24(24

)-en-3β-ol, an obtusifoliol-like molecule,

are also vesicle components in C. neoformans

(Rodrigues et al. 2007).

In addition to polysaccharides and lipids,

the extracellular vesicles of C. neoformans also

carried a complex array of proteins to the

extracellular milieu. Employing a proteomic

approach, 76 different proteins were identiﬁ ed as

vesicle components (Rodrigues et al. 2008).

Chaperones, including heat shock proteins and

superoxide dismutase, signal transduction regula-

tors, anti-oxidant and cytosolic proteins and

enzymes were identified. It is intriguing that

approximately one third of proteins identiﬁ ed in

the C. neoformans vesicles corresponded to

molecules that were previously described to

compose mammalian exosomes (Rodrigues

et al. 2008).

Vesicular Trans-Cell Wall Transport

in Histoplasma Capsulatum

and Other Fungal Species

Following the identiﬁ cation of extracellular vesi-

cles in the basidiomycetes C. neoformans, fungi

in the phylum Ascomycota were examined for

vesicle production. The ascomycetes H. capsula-

tum, C. albicans, C. parapsilosis, S. schenckii, and

S. cerevisiae each were found to generate hetero-

geneous extracellular vesicles (Albuquerque et al.

2008). Transmission electron microscopy studies

of supernatants subjected to fractional centrifuga-

tion revealed that each fungus produced extracel-

lular spherical, bilayered vesicles that varied in

their electron density and ranged in diameter from

10 to 350 nm. Vesicles were not present in super-

natants from media inoculated with dead fungal

cells. Notably, the percentage of large versus small

vesicles varied signiﬁ cantly between the different

ascomycetes. For example, only 4% of S. cerevi-

siae vesicles were larger than 50 nm compared to

38 and 54% of vesicles from H. capsulatum and

C. parapsilosis, respectively.

The movement of membrane-bound vesicles

across C. albicans cell walls was previously

proposed by Anderson and co-workers (Anderson

et al. 1990) who demonstrated the presence of

“pimples” in the cell wall of opaque switch variants

of strain WO-1 where vesicles were identiﬁ ed

within some channels or appeared to emerge from

the “pimples” with their double membrane intact.

The analysis of intact H. capsulatum yeast cells by

transmission electron microscopy similarly

demonstrated vesicular structures engaging the

internal aspects of the cell wall, within the cell

wall, and emerging from the cell wall consis-

tent with trans-cell wall transport of vesicles

(Albuquerque et al. 2008).

Trans-cell wall secretion in fungi

Lipid Insights 2008:2

To further define the constituents of the

ascomycetes vesicles, purified fractions from

H. capsulatum were subjected to lipidomic and

proteomic analyses (Albuquerque et al. 2008).

Using electrospray ionization time-of-ﬂ ight mass

spectrometry, phosphatidylethanolamine, phospha-

tidylserine (PS), and phosphatidylcholine (PC)

were identiﬁ ed as the major phospholipid species

comprising the vesicles, which is similar to the

typical distribution of lipids in pathogenic

yeast (Rattray et al. 1975). The protein compo-

nents of the vesicles were analyzed by cation

exchange chromatography and analyzed by liquid

chromatography-tandem mass spectrometry and

283 proteins were validated, with 206 proteins

identiﬁ ed by sequence analysis. Notably, several

proteins involved in H. capsulatum pathogenesis

and host immune responses were detected,

including chaperones (Hsp30, Hsp70, and Hsp60),

superoxide dismutase, and catalase B. Further-

more, proteins involved in signal transduction,

vesicle formation, cell wall and cytoskeleton

regulation, cell growth, and sugar, lipid and amino

acid metabolism were identiﬁ ed.

The fact that proteins associated with H. capsu-

latum virulence were identiﬁ ed in the puriﬁ ed

extracellular vesicles raised the question of whether

the vesicle proteins were recognized by the host

immune system. In this regard, hyperimmune

human serum reacted strongly with diverse vesic-

ular proteins, including Hsp60 and histone 2B.

Notably, H. capsulatum Hsp60 has been associated

with virulence (Allendoerfer et al. 1996; Deepe and

Gibbons, 2001; Deepe and Gibbons, 2002; Gomez

et al. 1995b; Scheckelhoff and Deepe, 2002) and

antibody to histone 2B can modify experimental

histoplasmosis (Nosanchuk et al. 2003). Hence, it

is likely that proteins transported via vesicles are

involved in host-pathogen interactions.

Putative Mechanisms of Vesicle

Diversity, Biogenesis and Passage

Through the Cell Wall

The ﬁ ndings of Yoneda and Doering (Yoneda and

Doering, 2006) suggesting that GXM-containing

vesicles in C. neoformans were derived from the

Golgi apparatus was further supported by Hu and

co-workers (Hu et al. 2007). These authors

reported that exposure of C. neoformans to

brefeldin A resulted in a massive reduction of

capsule size. The target of brefeldin A in mammalian

cells is a subset of Sec7-type GTP-exchange

factors that catalyze the activation of a small

GTPase called Arf1p (Nebenfuhr et al. 2002).

Arf1, in turn, is responsible for the recruitment

of coat proteins to membranes, resulting in the

formation of transport vesicles (Scales et al.

2000). Arf1p and brefeldin A-sensitive GTP-

exchange factors are localized to the Golgi

apparatus of mammalian and yeast cells (Spang

et al. 2001). Therefore, it seems clear that GXM

assembly at the cell surface requires the secretion

of post-Golgi vesicles ﬁ lled up with the polysac-

charide. These vesicles would be targeted to the

plasma membrane for exocytosis (Yoneda and

Doering, 2006), which would result in the release

of the polysaccharide to the periplasmic space.

Therefore, for capsule assembly, additional

mechanisms of trans-cell wall transport of GXM

would be required. However, a model of exocy-

tosis with discharge into the periplasmic space

did not account for transport of GXM across the

cell wall for assembly into a capsule in the

extracellular space.

Mutation of the SEC4 homolog that resulted in

the cytoplasmic accumulation of post-Golgi vesi-

cles did not lead to any apparent alteration in the

capsular expression phenotype (Yoneda and

Doering, 2006). This observation could be a

consequence of continued polysaccharide secretion

before the temperature shift, which blocks secre-

tion, or incomplete inhibition of the secretion

caused by the mutation. It was impossible to

distinguish in C. neoformans whether GXM was

targeted to the cell surface exclusively in post-

Golgi vesicles or via recycling endosomes. In fact,

endosomes and related multivesicular bodies

(MVBs) can be connected to the trans-Golgi secre-

tory pathway, so both pathways could be involved

in polysaccharide secretion in C. neoformans.

It is therefore possible that the post-golgi

GXM-enriched vesicles are sorted to another

compartment than the plasma membrane such as

the late endosomes and the MVBs.

The existence of MVB-like structures in

C. neoformans was suggested in early and recent

studies (Rodrigues et al. 2008; Takeo et al.

1973a, b) (Fig. 1). Some of these compartments

were found to be clearly merging with the plasma

membrane, indicating that the extracellular vesicles

of C. neoformans could be related to mammalian

exosomes. Exosomes are extracellular vesicles

derived from the fusion of MVBs with the plasma

Rodrigues et al

Lipid Insights 2008:2

membrane. Their isolation was extensively

described in several animal cell models such as

reticulocytes, dendritic cells, B lymphocytes,

mastocytes and epithelial cells (Johnstone et al.

1987; Lamparski et al. 2002; Raposo et al. 1996;

Skokos et al. 2003; van Niel et al. 2001).

The molecular machinery implied in MVB

formation and sorting is widely known in

S. cerevisiae (Hurley and Emr, 2006), but these

studies had never shown extracellular exosomes

in fungal cells. Early observations by Takeo and

colleagues (Takeo et al. 1973a) suggested the

occurrence of what the authors deﬁ ned as an “early

stage of formation of spherical invaginations which

secrete the vesicles outside the cell membrane”,

suggesting the occurrence of exosomes in

fungi. That supposition was strengthened by the

highly heterogeneous protein composition of

C. neoformans and H. capsulatum vesicles

(Albuquerque et al. 2008; Rodrigues et al. 2008).

This heterogeneous protein proﬁ le is also seen in

mammalian exosomes, indicating a possible

common origin for both extracellular vesicles.

Moreover, detergent-resistant lipid microdomains

are known to compose exosome membranes at

least in some mammalian cell types (de Gassart

et al. 2003). Lipid microdomains—or lipid

rafts – are mainly characterized by high sterol

content and presence of glycosphingolipids, as

described in fungal vesicles (Rodrigues et al.

2007). They are seemingly involved on the

sorting of exosomal proteins as well as in

vesicle morphology and formation (de Gassart

et al. 2003).

Laccase, an enzyme involved in melanin

synthesis, is another important component of

C. neoformans vesicles (Rodrigues et al. 2008).

Interestingly, it was recently shown that a

C. neoformans mutant defective in the vps34 gene

has a considerable decrease on melanin expression

(Hu et al. 2008). Vps34 (vacuolar protein sorting 34)

is involved in vesicular sorting of vacuolar hydro-

lases from the trans-Golgi-network to the late

endosomes and in the internal vesicle formation

within MVBs (Futter et al. 2001; Kihara et al.

2001). These data strongly support the idea that

laccase-containing vesicles originate from the

endocytic pathway.

CW PM

100 nm

MVB (?)

Figure 1. Typical MVB-like structures in fusion with the plasma membrane in C. neoformans. MVB internal vesicles (arrowheads) are released

outside the external layer of the plasma membrane, representing a potential mechanism of exosome biogenesis in fungi. This ﬁ gure was

originally published by Takeo and colleagues (Takeo et al. 1973b). Reproduced with permission from the American Society for Microbiology.

CW, cell wall; PM, plasma membrane; Cy, cytoplasm; MVB, multivesicular body.

Trans-cell wall secretion in fungi

Lipid Insights 2008:2

Although different lines of evidence point to a

relationship between fungal extracellular vesicles

and exosomes, other possibilities can not be ruled

out. The methods currently used for vesicle puri-

ﬁ cation do not discriminate between vesicles of

different origins, resulting in heterogeneous

preparations. Indeed, electron microscopy com-

bined to other approaches demonstrated that

vesicle fractions contain different subpopulations

which differ in morphological characteristics

(Rodrigues et al. 2008), suggesting the existence

of different intracellular compartments involved

in vesicle biogenesis (Fig. 2). Cellular vesicle

secretion strategies other than exosome formation

(Fig. 2B) could consist of simple membrane budding

(Fig. 2C) and other unconventional mechanisms

of vesicle release (Fig. 2D). The existence of

different mechanisms of vesicle biogenesis could,

in turn, result in the simultaneous generation of

extracellular membrane compartments of different

cellular origins.

Further investigations at the molecular and ultra-

structural levels will need to be done for the devel-

opment of reliable concepts of vesicle biogenesis.

In this context, the discovery of biochemical

markers for different vesicle sub-populations is

likely to be fundamental criterion for vesicular

identiﬁ cation and characterization. In this scenario,

genetic approaches will be powerful tools to under-

stand where the vesicles are assembled and how

they reach the extracellular space.

It is unknown how vesicles cross the cell wall to

reach the extracellular space. Transmission

electron microscopy revealed that the cell wall

of C. neoformans and H. capsulatum contain

numerous vesicle-like structures (Rodrigues et al.

2008; Albuquerque et al. 2008). The cell wall is now

described as a compact although malleable structure

Periplasm

cell wall

cytoplasm

Figure 2. Mechanisms for vesicle biogenesis in fungal cells. (A) Vesicles sorted to the cell surface by conventional secretion fuse with the

plasma membrane, releasing their cargo into the periplasm. MVB formation followed by fusion with the plasma membrane (B) would result

in the extracellular release of exosomes. Membrane budding (C) could also result in the periplasmic release of vesicles, as also proposed

for other unconventional secretion mechanisms (D). For conceptual clariﬁ cation, see (Kinseth et al. 2007) and (Keller et al. 2006). Of note,

vesicle content could vary according with biogenesis pathways. The possibility that different mechanisms result in the simultaneous generation

of extracellular vesicles cannot be ruled out.

Rodrigues et al

Lipid Insights 2008:2

(Nimrichter et al. 2005a), likely to suffer rearrangements,

that could allow vesicle passage. Indeed, it was

demonstrated by atomic force microscopy that the

cell wall of fungi contain pores that would allow

vesicle passage (de Souza Pereira and Geibel, 1999;

Eisenman et al. 2005). Trans-cell wall vesicle secre-

tion could also involve vesicle-mediated cell wall

remodeling. For example, H. capsulatum extracel-

lular vesicles contain enzymes regulating synthesis

and hydrolysis of cell wall components (Albuquerque

et al. 2008). In yeast cells, wall remodeling for

vesicle passage could be facilitated during budding,

when the cell wall is thinner in the bud than in non-

dividing areas of the cell surface (Linnemans et al.

1977). Finally, a myosin analog was described as a

cell wall component of Aspergillus fumigatus

(Esnault et al. 1999). In the cytosolic space, motor

proteins like myosin and dynein contribute with the

movement of transport vesicles (Schliwa and Woe-

hlke, 2003). However, it is completely unknown if

such proteins would be functional at the cell wall

microenvironment.

‘Virulence Bags’: Fungal Extracellular

Vesicles Concentrate Fungal

Molecules Involved in Pathogenesis

It is clear that fungal vesicles are released to the

extracellular milieu carrying a complex panel of

proteins and lipids and, in the case of C. neoformans,

polysaccharides (Rodrigues et al. 2007; Rodrigues

et al. 2008). Current data strongly suggest that vesicle

secretion occurs not only in vitro, but also in vivo

during animal infections by fungal pathogens.

Secreted vesicles were detected when murine mac-

rophages were incubated with C. neoformans yeasts

and also in sections of lung excised from mice infected

with this pathogen (Rodrigues et al. 2007). Vesicles

secreted during infection could directly mediate host

cell damage and/or modulation of immune response.

Indeed, similar functions were proposed for vesicles

released from the outer membrane of Gram negative

bacteria (Mashburn-Warren et al. 2008).

Lipid components of fungal vesicles have the

potential to modulate interactions of fungi with their

hosts. GlcCer is required for fungal growth in

alkaline pH and high CO

levels, as well as for the

virulence of C. neoformans (Rittershaus et al. 2006;

Saito et al. 2006). The fact that the vesicles secreted

by S. cerevisiae, which does not synthesize

GlcCer, are in a smaller size range (Albuquerque

et al. 2008) suggests an involvement of GlcCer in

vesicle assembly. As a vesicle-associated compound,

GlcCer could be constantly delivered to host cells,

facilitating the production of antimicrobial antibodies

(Rodrigues et al. 2000). In addition, lipid turnover

is known to be a well regulated phenomenon that

occurs in mammalian cells. Glycosphingolipids are

also involved with sorting of lipids and proteins in

mammalian cells (Degroote et al. 2004). Although

fungal and mammalian GlcCer are structurally dif-

ferent (Barreto-Bergter et al. 2004), secreted fungal

GlcCer could potentially interfere with recycling

and sorting of lipids and proteins in host cells. Other

lipids, including phosphatidylcholine, phosphatidyl-

ethanolamine and phosphatidylserine, were also

characterized in fungal vesicles (Albuquerque et al.

2008). Although these are common components of

biological membranes, their involvement in immune

responses has been already reported. Liposomes

carrying phosphatidylserine, for instance, can

modulate cytokine production, decrease microbial

killing and inhibit nitric oxide production by

macrophages (Aramaki, 2000; Gilbreath et al. 1986;

Hoffmann et al. 2005).

In bacteria, adhesins, toxins, and immunomodula-

tory compounds are the components of extracellular

vesicles, which directly mediate bacterial binding

and invasion, cause cytotoxicity, and modulate the

host immune response (Kuehn and Kesty, 2005).

In fungal vesicles, several proteins involved with

fungal pathogenesis were identiﬁ ed (Albuquerque

et al. 2008; Rodrigues et al. 2008). Concentration of

pathogenic determinants in the vesicles could provide

an efﬁ cient mechanism of release of virulence factors

into host tissues. Vesicle membranes could also

protect fungal structures against hydrolysis by host

extracellular enzymes. In addition, membrane fusion

involving vesicle elements and the surface of host

cells could result in the direct delivery of fungal

structures into host tissues.

Different immunogenic compounds are released

in fungal vesicles. Chaperones, including heat

shock proteins 60 and 70 from H. capsulatum and

C. neoformans, respectively, were detected in

extracellular vesicles by proteomics and serology

(Albuquerque et al. 2008; Rodrigues et al. 2008).

Hsp60 is responsible for binding of H. capsulatum

to the CD11/C18 receptor in human macrophages

(Long et al. 2003). This protein, which was initially

described as a glycoprotein present at membrane

and cell wall of H. capsulatum, confers protec-

tion against lethal intravenous challenge with

this pathogen (Gomez et al. 1995a). Besides

Trans-cell wall secretion in fungi

Lipid Insights 2008:2

representing an efﬁ cient vehicle for delivery of

immunogens during infection, vesicles carrying

Hsp60 could sequestrate anti-Hsp60 antibodies

produced by the host. In C. neoformans, the role

of Hsp70 is still unknown, but this protein is

considered to be a major immunogen in cryptococ-

cosis (Kakeya et al. 1997; Kakeya et al. 1999).

Pathogenic-related enzymes have also been

described as components of fungal extracellular

vesicles (Table 1) (Albuquerque et al. 2008;

Rodrigues et al. 2008). A number of proteins with

general anti-oxidant functions are also contained in

the extracellular vesicles, suggesting that vesicle

secretion could be beneﬁ cial for intracellular parasit-

ism and fungal persistence within host tissues.

Virulence factors of C. neoformans such as urease

and laccase were also vesicle associated (Rodrigues

et al. 2008). Interestingly, some of the vesicles had

electron dense structures consistent with pigments.

Laccase is responsible for melanin synthesis in

many fungal species, but it also interferes with

prostaglandin metabolism (Erb-Downward et al.

2008). Prostaglandins are lipid mediators that

regulate components of the immune response,

including defense against infection. For instance,

PGE

inhibits B and T lymphocyte proliferation, as

well as macrophage functions (Chouaib et al. 1985;

Simkin et al. 1987; Taffet et al. 1981; Taffet and

Russell, 1981; Xu et al, 2008). Prostaglandin produc-

tion by laccase present in vesicles has the potential

to negatively modulate the immune response.

As discussed in previous sections, cryptococcal

GXM is also secreted within vesicles that reach

the extracellular space. GXM is recognized by

different cell types and involved with a series of

immunomodulatory effects (Vecchiarelli, 2005).

Polysaccharide vesicular export, therefore, could

be not only a physiological solution for capsule

assembly, but also a fungal strategy to cause tissue

damage through the delivery of virulence factors

and defense proteins into host tissues. Interest-

ingly, two different enzymes with well-deﬁ ned

roles in capsule synthesis and virulence were also

found in cryptococcal vesicles (Rodrigues

et al. 2008).

Unsolved Problems and Future

Perspectives

The discovery of a trans-cell wall vesicular trans-

port system in C. neoformans, and its subsequent

extension to ascomycetous fungi, provides a

Table 1. Pathogenic determinants found in fungal extracellular vesicles.

Virulence

determinant

Function Organism

Catalases A and B Anti-oxidant defense C. neoformans (CatA)

and H. capsulatum (CatB)

Glucosylceramide Fungal growth C. neoformans

GXM Immunomodulation C. neoformans

Heat shock proteins Immunogen; adhesion

to host cells

C. neoformans

and H. capsulatum

Laccase Pigment synthesis;

prostaglandin metabolism

C. neoformans

Superoxide dismutase Anti-oxidant defense C. neoformans

and H. capsulatum

Thiol-speciﬁ c

antioxidant protein

Anti-oxidant defense C. neoformans

and H. capsulatum

Thioredoxin Anti-oxidant defense C. neoformans

Thioredoxin reductase Anti-oxidant defense C. neoformans

UDP-glucose

dehydrogenase

Conversion of UDP-glucose

into UDP-glucuronic acid

(capsule synthesis)

C. neoformans

UDP-glucuronic acid

decarboxylase

Converts UDP-glucuronic

acid to UDP-xylose

(capsule synthesis)

C. neoformans

Rodrigues et al

Lipid Insights 2008:2

solution to the problem of exporting macromole-

cules to the cellular exterior. The ﬁ nding of similar

vesicles in ascomycetes and basidiomycetes

suggest that the vesicular transport system is

ancient, such that its existence predated the diver-

gence of these fungal branches 0.5–1.0 billion

years ago. For C. neoformans the existence of such

an export mechanism would appear to be necessary

for capsular synthesis given this structure is

composed of macromolecules with mass that can

exceed 1 million Daltons (McFadden et al. 2006b).

The ﬁ nding that such vesicles contain numerous

fungal products associated with virulence suggests

that they also have a concentrative function that

could allow these compounds to function more

efﬁ ciently, and possibly synergistically. However,

the discovery of this trans-cell wall vesicular trans-

port system also poses a new set of questions for

future studies.

How many types of vesicles

are there?

Electron microscopy reveals that vesicles differ

in shape, size, and inter-vesicular contents.

EM surveys suggest the existence of at least four

types of vesicles in C. neoformans supernatants

(Rodrigues et al. 2008). Classifying vesicular

types is important because each type would sug-

gest the existence of a separate cellular synthetic

mechanism. Currently, vesicle types have been

discriminated based on morphology and size.

However, morphological criteria may not be

adequate to ascertain the various types of vesicles

since similar appearance does not necessarily

imply similar content. Consequently, continued

progress in this area will require the develop-

ment of methods for vesicular fractionation and

chemical characterization. Furthermore, a full

description of the vesicular transport system will

require a correlation of vesicular content with

morphological characteristics, which is likely to

involve combined proteomic analysis, serological

reactivity, enzymatic assays, etc.

Where do vesicles originate from?

Analysis of protein components and the ﬁ nding of

vesicles within vacuolar structures led to the

proposal that extracellular vesicles originate in

exosome-like structures (Rodrigues et al. 2008).

However, such structures have yet to be described

in fungi. Deﬁ nition of the various vesicle types

combined with mutational analysis could be used to

ascertain the number of vesicular synthetic pathways

in fungal cells. Understanding the sites of vesicular

synthesis and the relationship of extracellular vesi-

cles to cytoplasmic vesicles will undoubtedly be a

major area of investigation in future years.

How are vesicles transported across

the cell wall?

There is no information on the mechanism used to

shuttle vesicles across the cell wall. The existence

of pores on the cell wall with dimensions that

approximate those of vesicles suggest that these

could be used for vesicular transport. Vesicular

transport across pores raises a variety of other

questions including the energy dynamics of such

processes and the types of motors and gears that

would be needed to shuttle such large structures

across rigid cell walls. This is made even more

complex when one considers that certain fungi,

such as C. neoformans, H. capsulatum and

S. schenckii can form dense melanin layers within

their cell walls (for review, (Nosanchuk and

Casadevall, 2006)).

What is the fate of released vesicles?

In the case of C. neoformans, vesicular transport

has been proposed as the mechanism to deliver

macromolecules for capsular assembly to the exte-

rior of the cell (Rodrigues et al. 2007). If that is

the case, it raises the question of how vesicles are

directed to the capsule surface and their cargo

unloaded. Extracellular phospholipases may

degrade phospholipids membranes and allow cargo

discharge. C. neoformans and most pathogenic

fungi have extracellular phospholipase activity

(Chayakulkeeree et al. 2008; Ghannoum, 1998;

Hruskova-Heidingsfeldova, 2008; Simockova

et al. 2008). A related issue is the relative paucity

of vesicles relative to the number of cells in solu-

tion. This could be interpreted as indicating that

vesicles are relatively short lived structures that

release their contents in the extracellular space. It

is possible that vesicles recovered from superna-

tants reﬂ ect a very small minority of the secreted

vesicles and that isolation of the overwhelming

majority of vesicles produced by fungal cells would

require the development of more sophisticated

recovery techniques. Hence, understanding the

Trans-cell wall secretion in fungi

Lipid Insights 2008:2

dynamics of vesicular transport will require studies

on the fate of released vesicles.

What is the relationship between

vesicles and host cell toxicity?

C. neoformans intracellular pathogenesis is

characterized by the appearance of numerous

vesicles in the cytoplasm of phagocytic host cells

that contain cryptococcal capsular polysaccharide

(Feldmesser et al. 2000). These vesicles are

believed to be toxic to the host cells since their

accumulation is temporally correlated with cyto-

toxic changes that include membrane blebs and

phagosomal membrane leakiness (Feldmesser et al.

2000; Tucker and Casadevall, 2002). The discovery

of a vesicular transport system for C. neoformans

raises the possibility that these cytoplasmic vesicles

originated as fungal vesicles that were released from

yeast-containing phagosomes. Hence, knowing

about the effect of shed vesicles on host cells and

their relationship to the polysaccharide-containing

vesicles that accumulate in host cells would seem

to be an important question for understanding the

pathogenesis of cryptococcal infections. Further-

more, it is possible that shed vesicles are also

involved in the pathogenesis of other facultative

intracellular fungi such as H. capsulatum.

What is the internal organization

of vesicular components?

Given that vesicles contain multiple fungal compo-

nents that include lipids, polysaccharides, carbohy-

drates and proteins, it is possible that these are

packaged inside vesicles in an organized manner that

could reflect their addition following or during

vesicular synthesis. For example, it is conceivable

that vesicles destined for capsular synthesis contain

capsular components arranged in a manner that

facilitates their function. Understanding the packaging

characteristics of vesicles is likely to be an essential

aspect of understanding their function and mecha-

nisms of action. Such studies will almost certainly

require the application of new microscopy techniques

in combination with speciﬁ c reagents, such as mono-

clonal antibodies, that delineate the position of spe-

ciﬁ c components within the vesicular structure.

Finally, there is the notion that knowledge of

vesicular transport systems may be exploited in

the design of new types of antifungal drugs and

vaccines. The complex choreography of vesicular

transport implies the existence of numerous steps

that could be targets of drug development. One could

imagine drugs that reduce the virulence of fungi by

interfering with vesicular synthesis and transport.

Identiﬁ cation of common pathways used by differ-

ent pathogenic fungi for vesicular transport could

lead to the design of broad-spectrum antifungal

drugs. Since trans-cell wall vesicular transport

mechanisms have no counterpart on mammalian

cells such drugs could potentially have high speci-

ﬁ city for fungal cells while concomitantly having

limited host cells toxicity. Furthermore, it is possible

that vesicular preparations can be developed into

vaccine formulations. The ﬁ nding of many fungal

components associated with virulence in vesicles

and the fact that vesicle-related proteins are immu-

nogenic (Albuquerque et al. 2008; Rodrigues et al.

2008) suggest that vesicle preparations and/or their

components could have utility as fungal vaccines.

In this regard, it is noteworthy that exosome prepa-

rations from dendritic cells are been investigated as

potential anti-cancer vaccines (Hao et al. 2007).

It is now apparent that the discovery of vesicles in

cryptococcal culture supernatants has opened a new

window into fungal cell biology that promises to

revolutionize our thinking on fungal product export

and its impact on pathogenesis. It is also clearly appar-

ent that this area of investigation is extremely complex

and will require the development of new experimen-

tal tools and reagents for continued progress.

Acknowledgements

MLR and LN are supported by grants from the

Brazilian agencies FAPERJ and CNPq. AC is

supported by NIH grants AI033142, AI033774,

AI052733, and HL059842. JDN is supported by

NIH AI52733 and AI056070-01A2. We are

thankful to Igor C. Almeida, Rosana Puccia and

Luiz R. Travassos for helpful discussions.

Disclosure

The authors report no conﬂ icts of interest.

References

Albuquerque, P.C., Nakayasu, E.S., Rodrigues, M.L., Frases, S.,

Casadevall, A., Zancope-Oliveira, R.M., Almeida, I.C. and

Nosanchuk, J.D. 2008. Vesicular transport in Histoplasma capsulatum:

an effective mechanism for trans-cell wall transfer of proteins and

lipids in ascomycetes. Cell. Microbiol.

Allendoerfer, R., Maresca, B. and Deepe, G.S., Jr., 1996. Cellular immune

responses to recombinant heat shock protein 70 from Histoplasma

capsulatum. Infect. Immun., 64:4123–8.

Rodrigues et al

Lipid Insights 2008:2

Anderson, J., Mihalik, R. and Soll, D.R. 1990. Ultrastructure and antigenicity

of the unique cell wall pimple of the Candida opaque phenotype.

J. Bacteriol., 172:224–35.

Aramaki, Y. 2000. Liposomes as immunomodulator—inhibitory effect of

liposomes on NO production from macrophages. Biol. Pharm. Bull.,

23:1267–74.

Barreto-Bergter, E., Pinto, M.R. and Rodrigues, M.L. 2004. Structure and

biological functions of fungal cerebrosides. An Acad. Bras. Cienc.,

76:67–84.

Bingle, L.E., Bailey, C.M. and Pallen, M.J. 2008. Type VI secretion: a

beginner’s guide. Curr. Opin. Microbiol., 11:3–8.

Chayakulkeeree, M., Sorrell, T.C., Siafakas, A.R., Wilson, C.F., Pantarat, N.,

Gerik, K.J., Boadle, R. and Djordjevic, J.T. 2008. Role and

Mechanism of Phosphatidylinositol-Speciﬁ c Phospholipase C in

Survival and Virulence of Cryptococcus neoformans. Mol.

Microbiol.

Chouaib, S., Welte, K., Mertelsmann, R. and Dupont, B. 1985. Prostaglandin

E2 acts at two distinct pathways of T lymphocyte activation: inhibi-

tion of interleukin 2 production and down-regulation of transferrin

receptor expression. J. Immunol., 135:1172–9.

Cianciotto, N.P. 2005. Type II secretion: a protein secretion system for all

seasons. Trends Microbiol., 13:581–8.

Craig, L. and Li, J. 2008. Type IV pili: paradoxes in form and function.

Curr. Opin. Struct. Biol., 18:267–77.

Davidson, A.L. and Maloney, P.C. 2007. ABC transporters: how small

machines do a big job. Trends Microbiol., 15:448–55.

de Gassart, A., Geminard, C., Fevrier, B., Raposo, G. and Vidal, M. 2003.

Lipid raft-associated protein sorting in exosomes. Blood,

102:4336–44.

de Souza Pereira, R. and Geibel, J. 1999. Direct observation of oxidative

stress on the cell wall of Saccharomyces cerevisiae strains with atomic

force microscopy. Mol. Cell Biochem., 201:17–24.

Deepe, G.S., Jr., and Gibbons, R. 2001. V beta 6+ T cells are obligatory for

vaccine-induced immunity to Histoplasma capsulatum. J. Immunol.,

167:2219–26.

Deepe, G.S., Jr., and Gibbons, R.S. 2002. Cellular and molecular regulation

of vaccination with heat shock protein 60 from Histoplasma

capsulatum. Infect. Immun., 70:3759–67.

Degroote, S., Wolthoorn, J. and van Meer, G. 2004. The cell biology of

glycosphingolipids. Semin. Cell. Dev. Biol., 15:375–87.

Eisenman, H.C., Nosanchuk, J.D., Webber, J.B., Emerson, R.J., Camesano,

T.A. and Casadevall, A. 2005. Microstructure of cell wall-associated

melanin in the human pathogenic fungus Cryptococcus neoformans.

Biochemistry, 44:3683–93.

Engel, L.S., Hill, J.M., Caballero, A.R., Green, L.C. and O’Callaghan, R.J.

1998. Protease IV, a unique extracellular protease and virulence fac-

tor from Pseudomonas aeruginosa. J. Biol. Chem., 273:16792–7.

Erb-Downward, J.R., Noggle, R.M., Williamson, P.R. and Huffnagle, G.B.

2008. The role of laccase in prostaglandin production by Cryptococcus

neoformans. Mol. Microbiol., 68:1428–37.

Esnault, K., el Moudni, B., Bouchara, J.P., Chabasse, D. and Tronchin, G.

1999. Association of a myosin immunoanalogue with cell envelopes

of Aspergillus fumigatus conidia and its participation in swelling and

germination. Infect. Immun., 67:1238–44.

Feldmesser, M., Kress, Y. and Casadevall, A. 2001. Dynamic changes in

the morphology of Cryptococcus neoformans during murine

pulmonary infection. Microbiology, 147:2355–65.

Feldmesser, M., Kress, Y., Novikoff, P. and Casadevall, A. 2000.

Cryptococcus neoformans is a facultative intracellular pathogen in

murine pulmonary infection. Infect. Immun., 68:4225–37.

Futter, C.E., Collinson, L.M., Backer, J.M. and Hopkins, C.R. 2001. Human

VPS34 is required for internal vesicle formation within multivesicular

endosomes. J. Cell Biol., 155:1251–64.

Garcia-Rivera, J., Chang, Y.C., Kwon-Chung, K.J. and Casadevall, A. 2004.

Cryptococcus neoformans CAP59 (or Cap59p) is involved in the

extracellular trafﬁ cking of capsular glucuronoxylomannan.

Eukaryot.

Cell, 3:385–92.

Ghannoum, M.A. 1998. Extracellular phospholipases as universal virulence

factor in pathogenic fungi. Nippon Ishinkin Gakkai Zasshi,

39:55–9.

Gilbreath, M.J., Hoover, D.L., Alving, C.R., Swartz, G.M., Jr., and

Meltzer, M.S. 1986. Inhibition of lymphokine-induced macrophage

microbicidal activity against Leishmania major by liposomes:

characterization of the physicochemical requirements for liposome

inhibition. J. Immunol., 137:1681–7.

Glick, B.S. and Malhotra, V. 1998. The curious status of the Golgi apparatus.

Cell., 95:883–9.

Gomez, F.J., Allendoerfer, R. and Deepe, G.S., Jr., 1995a. Vaccination with

recombinant heat shock protein 60 from Histoplasma capsulatum

protects mice against pulmonary histoplasmosis. Infect. Immun.,

63:2587–95.

Gomez, F.J., Allendoerfer, R. and Deepe, G.S., Jr., 1995b. Vaccination with

recombinant heat shock protein 60 from Histoplasma capsulatum

protects mice against pulmonary histoplasmosis. Infect. Immun.,

63:2587–95.

Gorelick, F.S. and Shugrue, C. 2001. Exiting the endoplasmic reticulum.

Mol. Cell Endocrinol., 177:13–8.

Hao, S., Moyana, T. and Xiang, J. 2007. Review: cancer immunotherapy

by exosome-based vaccines. Cancer Biother. Radiopharm.,

22:692–703.

Heath, I.B., Gay, J.L. and Greenwood, A.D. 1971. Cell wall formation in

the Saprolegiales: cytoplasmicvesicles underlying developing walls.

Henderson, I.R., Navarro-Garcia, F., Desvaux, M., Fernandez, R.C. and

Ala’Aldeen, D. 2004. Type V protein secretion pathway: the

autotransporter story. Microbiol. Mol. Biol. Rev., 68:692–744.

Hoegl, L., Ollert, M. and Korting, H.C. 1996. The role of Candida albicans

secreted aspartic proteinase in the development of candidoses. J. Mol.

Med., 74:135–42.

Hoffmann, P.R., Kench, J.A., Vondracek, A., Kruk, E., Daleke, D.L.,

Jordan, M., Marrack, P., Henson, P.M. and Fadok, V.A. 2005. Interaction

between phosphatidylserine and the phosphatidylserine receptor

inhibits immune responses in vivo. J. Immunol., 174:1393–404.

Hruskova-Heidingsfeldova, O. 2008. Secreted proteins of Candida albicans.

Front Biosci., 13:7227–42.

Hu, G., Hacham, M., Waterman, S.R., Panepinto, J., Shin, S., Liu, X.,

Gibbons, J., Valyi-Nagy, T., Obara, K., Jaffe, H.A., Ohsumi, Y. and

Williamson, P.R. 2008. PI3K signaling of autophagy is required for

starvation tolerance and virulenceof Cryptococcus neoformans.

J. Clin. Invest., 118:1186–97.

Hu, G., Steen, B.R., Lian, T., Sham, A.P., Tam, N., Tangen, K.L. and

Kronstad, J.W. 2007. Transcriptional regulation by protein kinase

A in Cryptococcus neoformans. PLoS Pathog., 3:e42.

Hube, B. 1996. Candida albicans secreted aspartyl proteinases. Curr. Top

Med. Mycol., 7:55–69.

Hurley, J.H. and Emr, S.D. 2006. The ESCRT complexes: structure and

mechanism of a membrane-trafﬁ cking network. Annu. Rev. Biophys.

Biomol. Struct., 35:277–98.

Johnstone, R.M., Adam, M., Hammond, J.R., Orr, L. and Turbide, C. 1987.

Vesicle formation during reticulocyte maturation. Association of

plasma membrane activities with released vesicles (exosomes).

J. Biol. Chem., 262:9412–20.

Kakeya, H., Udono, H., Ikuno, N., Yamamoto, Y., Mitsutake, K., Miyazaki,

T., Tomono, K., Koga, H., Tashiro, T., Nakayama, E. and Kohno, S.

1997. A 77-kilodalton protein of Cryptococcus neoformans, a member

of the heat shock protein 70 family, is a major antigen detected in the

sera of mice with pulmonary cryptococcosis. Infect. Immun.,

65:1653–8.

Kakeya, H., Udono, H., Maesaki, S., Sasaki, E., Kawamura, S., Hossain, M.A.,

Yamamoto, Y., Sawai, T., Fukuda, M., Mitsutake, K., Miyazaki, Y.,

Tomono, K., Tashiro, T., Nakayama, E. and Kohno, S. 1999. Heat

shock protein 70 (hsp70) as a major target of the antibody response

in patients with pulmonary cryptococcosis. Clin. Exp. Immunol.,

115:485–90.

J. Gen. Microbiol., 65:225–232.

Trans-cell wall secretion in fungi

Lipid Insights 2008:2

Keller, S., Sanderson, M.P., Stoeck, A. and Altevogt, P. 2006. Exosomes:

from biogenesis and secretion to biological function. Immunol. Lett.,

107:102–8.

Kihara, A., Noda, T., Ishihara, N. and Ohsumi, Y. 2001. Two distinct Vps34

phosphatidylinositol 3-kinase complexes function in autophagy and

carboxypeptidase Y. sorting in Saccharomyces cerevisiae. J. Cell

Biol., 152:519–30.

Kinseth, M.A., Anjard, C., Fuller, D., Guizzunti, G., Loomis, W.F. and

Malhotra, V. 2007. The Golgi-associated protein GRASP is required

for unconventional protein secretion during development. Cell,

130:524–34.

Kuehn, M.J. and Kesty, N.C. 2005. Bacterial outer membrane vesicles and

the host-pathogen interaction. Genes Dev., 19:2645–55.

Lamparski, H.G., Metha-Damani, A., Yao, J.Y., Patel, S., Hsu, D.H.,

Ruegg, C. and Le Pecq, J.B. 2002. Production and characterization

of clinical grade exosomes derived from dendritic cells. J. Immunol.

Methods, 270:211–26.

Lee, M.C., Miller, E.A., Goldberg, J., Orci, L. and Schekman, R. 2004.

Bi-directional protein transport between the ER. and Golgi. Annu.

Rev. Cell Dev. Biol., 20:87–123.

Linnemans, W.A., Boer, P. and Elbers, P.F. 1977. Localization of acid

phosphatase in Saccharomyces cerevisiae: a clue to cell wall

formation. J. Bacteriol., 131:638–44.

Liu, G.Y. and Nizet, V. 2004. Extracellular virulence factors of group B.

Streptococci. Front Biosci., 9:1794–802.

Long, K.H., Gomez, F.J., Morris, R.E. and Newman, S.L. 2003. Identiﬁ ca-

tion of heat shock protein 60 as the ligand on Histoplasma capsulatum

that mediates binding to CD18 receptors on human macrophages.

J. Immunol., 170:487–94.

Mashburn-Warren, L., McLean, R.J. and Whiteley, M. 2008. Gram-negative

outer membrane vesicles: beyond the cell surface. Geobiology,

6:214–9.

Mazar, J. and Cotter, P.A. 2007. New insight into the molecular mechanisms

of two-partner secretion. Trends Microbiol., 15:508–15.

McClelland, E.E., Bernhardt, P. and Casadevall, A. 2005. Coping with multiple

virulence factors: which is most important? PLoS Pathog., 1:e40.

McFadden, D., Zaragoza, O. and Casadevall, A. 2006a. The capsular dynamics

of Cryptococcus neoformans. Trends Microbiol., 14:497–505.

McFadden, D.C., De Jesus, M. and Casadevall, A. 2006b. The physical

properties of the capsular polysaccharides from Cryptococcus

neoformans suggest features for capsule construction. J. Biol. Chem.,

281:1868–75.

Mitchell, A.P. 2006. Cryptococcal virulence: beyond the usual suspects.

J. Clin. Invest, 116:1481–3.

Mori, H. and Ito, K. 2001. The Sec protein-translocation pathway. Trends

Microbiol., 9:494–500.

Nebenfuhr, A., Ritzenthaler, C. and Robinson, D.G. 2002. Brefeldin A:

deciphering an enigmatic inhibitor of secretion. Plant Physiol.,

130:1102–8.

Nickel, W. and Seedorf, M. 2008. Unconventional Mechanisms of Protein

Transport to the Cell. Surface of Eukaryotic Cells. Annu. Rev. Cell

Dev. Biol.,

Niimi, M., Tanabe, K., Wada, S., Yamazaki, A., Uehara, Y., Niimi, K.,

Lamping, E., Holmes, A.R., Monk, B.C. and Cannon, R.D. 2005.

[ABC transporters of pathogenic fungi: recent advances in functional

analyses]. Nippon Ishinkin Gakkai Zasshi, 46:249–60.

Nimrichter, L., Cerqueira, M.D., Leitao, E.A., Miranda, K., Nakayasu, E.

S., Almeida, S.R., Almeida, I.C., Alviano, C.S., Barreto-Bergter, E.

and Rodrigues, M.L. 2005a. Structure, cellular distribution,

antigenicity, and biological functions of Fonsecaea pedrosoi ceramide

monohexosides. Infect. Immun., 73:7860–8.

Nimrichter, L., Rodrigues, M.L., Rodrigues, E.G. and Travassos, L.R. 2005b.

The multitude of targets for the immune system and drug therapy in

the fungal cell wall. Microbes. Infect., 7:789–98.

Nosanchuk, J.D. and Casadevall, A. 2006. Impact of melanin on microbial

virulence and clinical resistance to antimicrobial compounds.

Antimicrob. Agents Chemother., 50:3519–28.

Nosanchuk, J.D., Steenbergen, J.N., Shi, L., Deepe, G.S., Jr., and Casadevall,

A. 2003. Antibodies to a cell surface histone-like protein protect

against Histoplasma capsulatum. J. Clin. Invest., 112:1164–75.

Novick, P., Field, C. and Schekman, R. 1980. Identification of

23 complementation groups required for post-translational events in

the yeast secretory pathway. Cell., 21:205–15.

Novick, P. and Schekman, R. 1979. Secretion and cell-surface growth are

blocked in a temperature-sensitive mutant of Saccharomyces

cerevisiae. Proc. Natl. Acad. Sci. U.S.A., 76:1858–62.

Raposo, G., Nijman, H.W., Stoorvogel, W., Liejendekker, R., Harding, C.V.,

Melief, C.J. and Geuze, H.J. 1996. B. lymphocytes secrete antigen-

presenting vesicles. J. Exp. Med., 183:1161–72.

Rattray, J.B., Schibeci, A. and Kidby, D.K. 1975. Lipids of yeasts. Bacteriol

Rev., 39:197–231.

Rigel, N.W. and Braunstein, M. 2008. A new twist on an old pathway—accessory

secretion systems. Mol. Microbiol.,

Rittershaus, P.C., Kechichian, T.B., Allegood, J.C., Merrill, A.H., Jr.,

Hennig, M., Luberto, C. and Del Poeta, M. 2006. Glucosylceramide

synthase is an essential regulator of pathogenicity of Cryptococcus

neoformans. J. Clin. Invest., 116:1651–9.

Rodrigues, M.L., Nakayasu, E.S., Oliveira, D.L., Nimrichter, L.,

Nosanchuk, J.D., Almeida, I.C. and Casadevall, A. 2008. Extracellular

vesicles produced by Cryptococcus neoformans contain protein

components associated with virulence. Eukaryot. Cell., 7:58–67.

Rodrigues, M.L., Nimrichter, L., Oliveira, D.L., Frases, S., Miranda, K.,

Zaragoza, O., Alvarez, M., Nakouzi, A., Feldmesser, M. and

Casadevall, A. 2007. Vesicular polysaccharide export in Cryptococcus

neoformans is a eukaryotic solution to the problem of fungal trans-cell

wall transport. Eukaryot. Cell., 6:48–59.

Rodrigues, M.L., Travassos, L.R., Miranda, K.R., Franzen, A.J., Rozental,

S., de Souza, W., Alviano, C.S. and Barreto-Bergter, E. 2000. Human

antibodies against a puriﬁ ed glucosylceramide from Cryptococcus

neoformans inhibit cell budding and fungal growth. Infect. Immun.,

68:7049–60.

Saier, M.H., Jr., 2006. Protein secretion and membrane insertion systems

in gram-negative bacteria. J. Membr. Biol., 214:75–90.

Saito, K., Takakuwa, N., Ohnishi, M. and Oda, Y. 2006. Presence of gluco-

sylceramide in yeast and its relation to alkali tolerance of yeast. Appl.

Microbiol. Biotechnol., 71:515–21.

Salama, N.R. and Schekman, R.W. 1995. The role of coat proteins in the

biosynthesis of secretory proteins. Curr. Opin. Cell Biol., 7:536–43.

Sargent, F. 2007. The twin-arginine transport system: moving folded proteins

across membranes. Biochem. Soc. Trans., 35:835–47.

Scales, S.J., Gomez, M. and Kreis, T.E. 2000. Coat proteins regulating

membrane trafﬁ c. Int. Rev. Cytol., 195:67–144.

Scheckelhoff, M. and Deepe, G.S., Jr., 2002. The protective immune response

to heat shock protein 60 of Histoplasma capsulatum is mediated by

a subset of V beta 8.1/8.2+ T cells. J. Immunol., 169:5818–26.

Schekman, R. 2002. Lasker Basic Medical Research Award. SEC mutants

and the secretory apparatus. Nat. Med., 8:1055–8.

Schekman, R., Esmon, B., Ferro-Novick, S., Field, C. and Novick, P. 1983.

Yeast secretory mutants: isolation and characterization. Methods

Enzymol., 96:802–15.

Schekman, R. and Novick, P. 2004. 23 genes, 23 years later. Cell., 116:

S13–5,1pfollowingS9.

Schliwa, M. and Woehlke, G. 2003. Molecular motors. Nature,

422:759–65.

Simkin, N.J., Jelinek, D.F. and Lipsky, P.E. 1987. Inhibition of human B. cell

responsiveness by prostaglandin E2. J. Immunol., 138:1074–81.

Simockova, M., Holic, R., Tahotna, D., Patton-Vogt, J. and Griac, P. 2008.

Yeast Pgc1p (YPL206c) controls the amount of phosphatidylglycerol

via a phospholipase C-type degradation mechanism. J. Biol. Chem.,

283:17107–15.

Skokos, D., Botros, H.G., Demeure, C., Morin, J., Peronet, R., Birkenmeier,

G., Boudaly, S. and Mecheri, S. 2003. Mast cell-derived exosomes

induce phenotypic and functional maturation of dendritic cells and elicit

speciﬁ c immune responses in vivo. J. Immunol., 170:3037–45.

Rodrigues et al

Lipid Insights 2008:2

Spang, A., Herrmann, J.M., Hamamoto, S. and Schekman, R. 2001. The ADP

ribosylation factor-nucleotide exchange factors Gea1p and Gea2p have

overlapping, but not redundant functions in retrograde transport from

the Golgi to the endoplasmic reticulum. Mol. Biol. Cell., 12:1035–45.

Taffet, S.M., Pace, J.L. and Russell, S.W. 1981. Lymphokine maintains

macrophage activation for tumor cell killing by interfering with the

negative regulatory effect of prostaglandin E2. J. Immunol., 127:121–4.

Taffet, S.M. and Russell, S.W. 1981. Macrophage-mediated tumor cell

killing: regulation of expression of cytolytic activity by prostaglandin

E. J. Immunol., 126:424–7.

Takeo, K., Uesaka, I., Uehira, K. and Nishiura, M. 1973a. Fine structure

of Cryptococcus neoformans grown in vitro as observed by

freeze-etching. J. Bacteriol., 113:1442–8.

Takeo, K., Uesaka, I., Uehira, K. and Nishiura, M. 1973b. Fine structure

of Cryptococcus neoformans grown in vivo as observed by

freeze-etching. J. Bacteriol., 113:1449–54.

Tucker, S.C. and Casadevall, A. 2002. Replication of Cryptococcus neoformans

in macrophages is accompanied by phagosomal permeabilization and

accumulation of vesicles containing polysaccharide in the cytoplasm.

Proc. Natl. Acad. Sci. U.S.A., 99:3165–70.

van Niel, G., Raposo, G., Candalh, C., Boussac, M., Hershberg, R.,

Cerf-Bensussan, N. and Heyman, M. 2001. Intestinal epithelial cells

secrete exosome-like vesicles. Gastroenterology, 121:337–49.

Vecchiarelli, A. 2005. The cellular responses induced by the capsular poly-

saccharide of Cryptococcus neoformans differ depending on the

presence or absence of speciﬁ c protective antibodies. Curr. Mol.

Med., 5:413–20.

Veenendaal, A.K., van der Does, C. and Driessen, A.J. 2004.

The protein-conducting channel SecYEG. Biochim. Biophys. Acta.,

1694:81–95.

Walworth, N.C., Goud, B., Kabcenell, A.K. and Novick, P.J. 1989.

Mutational analysis of SEC4 suggests a cyclical mechanism for the

regulation of vesicular trafﬁ c. EMBO J., 8:1685–93.

Whitﬁ eld, C. 2006. Biosynthesis and assembly of capsular polysaccharides

in Escherichia coli. Annu. Rev. Biochem., 75:39–68.

Whitﬁ eld, C., Kaniuk, N. and Frirdich, E. 2003. Molecular insights into the

assembly and diversity of the outer core oligosaccharide in lipopoly-

saccharides from Escherichia coli and Salmonella. J. Endotoxin Res.,

9:244–9.

Wickner, W. and Schekman, R. 2005. Protein translocation across biological

membranes. Science, 310:1452–6.

Xu, X.J., Reichner, J.S., Mastrofrancesco, B., Henry, W.L., Jr., and Albina,

J.E. 2008. Prostaglandin E2 suppresses lipopolysaccharide-stimulated

IFN-beta production. J. Immunol., 180:2125–31.

Yoneda, A. and Doering, T.L. 2006. A eukaryotic capsular polysaccharide

is synthesized intracellularly and secreted via exocytosis. Mol. Biol.

Cell., 17:5131–40.

Zaragoza, O., Telzak, A., Bryan, R.A., Dadachova, E. and Casadevall, A.

2006. The polysaccharide capsule of the pathogenic fungus

Cryptococcus neoformans enlarges by distal growth and is rearranged

during budding. Mol. Microbiol., 59:67–83.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and

metabolism in the human pathogen Cryptococcus neoformans

Lívia Kmetzsch

, Charley Christian Staats

a,b

, Elisa Simon

, Fernanda L. Fonseca

, Débora L. Oliveira

Luna S. Joffe

, Jéssica Rodrigues

, Rogério F. Lourenço

, Suely L. Gomes

, Leonardo Nimrichter

Marcio L. Rodrigues

, Augusto Schrank

a,b

, Marilene Henning Vainstein

a,b,

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, 43421, Caixa Postal 15005, Porto Alegre, RS 91501-970, Brazil

Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Brazil

Laboratório de Estudos Integrados em Bioquímica Microbiana, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas

Filho 373, CCS, Bloco I. Rio de Janeiro, RJ, 21941-902, Brazil

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Professor Lineu Prestes, 748, 05508-900, São Paulo, SP, Brazil

a r t i c l e i n f o

Article history:

Received 7 June 2010

Accepted 22 July 2010

Available online xxxx

Keywords:

Nitrogen metabolism

Nitrogen Catabolite Repression

Cryptococcus neoformans

a b s t r a c t

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of

zinc ﬁnger containing transcriptional regulators known as GATA-factors. These factors activate the tran-

scription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are

absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Can-

dida albicans GAT1 genes. In an attempt to deﬁne the function of this transcriptional regulator in C. neo-

formans, we generated null mutants (gat1

) of this gene. The gat1 mutant exhibited impaired growth on

all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore,

the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently

mimics a low-quality nitrogen source. Gat1 is not required for C. neoformans survival during macrophage

infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identiﬁca-

tion of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitro-

gen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule

biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans.

1. Introduction

Pathogenic fungi have to adapt and survive in distinct nutritive

environments and, in this context, nitrogen uptake and its metab-

olism are critical to fungal growth. Nitrogen Catabolite Repression

(NCR) is a mechanism that controls the fungi selective utilization

of optimal nitrogen sources in preference to poor ones (

Marzluf,

1997). NCR-sensitive genes are repressed when preferred nitrogen

sources are available. Under limitation of these sources, the

expression of genes encoding permeases and catabolic enzymes,

required to utilize poor nitrogen sources, is activated by speciﬁc

GATA-factor family of transcription factors. Members of this family

contain a zinc-ﬁnger domain and are conserved in fungi (

Coffman

and Cooper, 1997; Coffman et al., 1996; Magasanik and Kaiser,

2002; Marzluf, 1997

). In Saccharomyces cerevisiae, two GATA-fac-

tors involved in activation of NCR-sensitive genes (Gat1 and

Gln3) were described (

Stanbrough et al., 1995). The GATA-type

transcriptional activator Gat1 and Gln3 orthologs of Candida albi-

cans regulate nitrogen metabolism and virulence during host-path-

ogen interactions (Liao et al., 2008; Limjindaporn et al., 2003). The

knockout of GAT1 in C. albicans results in reduced capacity to

metabolize some secondary nitrogen sources, but dimorphism is

not impaired (

Limjindaporn et al., 2003). The ortholog of GAT1 in

Aspergillus fumigatus (AreA) also participates in nitrogen regulation

and virulence, since null mutants for AreA show attenuated viru-

lence in a murine model of pulmonary aspergillosis (

Hensel

et al., 1998

Exposure of S. cerevisiae cells to rapamycin mimics a low-qual-

ity nitrogen source, which results in activation of NCR-sensitive

genes by Gat1 and Gln3 transcription factors (

Hardwick et al.,

1999; Scherens et al., 2006

). This immunosuppressant drug inhib-

its a conserved signaling cascade for cell proliferation regulated by

target of rapamycin (TOR) in response to nutrient availability

(

Jiang and Broach, 1999; Thomas and Hall, 1997). S. cerevisiae

Gln3 and Gat1 are phosphorylated in a Tor-dependent mechanism,

resulting in interaction with the cytoplasmic protein Ure2. Upon

rapamycin treatment, Gln3 and Gat1 are dephosphorylated,

doi:

10.1016/j.fgb.2010.07.011

* Corresponding author at: Centro de Biotecnologia, Universidade Federal do Rio

Grande do Sul, Avenida Bento Gonçalves 9500, 43421, Setor 4, Porto Alegre, RS

91501-970, Brazil. Fax: +55 51 3308 7309.

E-mail address:

[email protected] (M.H. Vainstein).

Fungal Genetics and Biology xxx (2010) xxx–xxx

Contents lists available at ScienceDirect

Fungal Genetics and Biology

jo u rn a l h om ep a ge : w w w. e ls ev i er . co m /l o ca te / yf g bi

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

released and directed to the nucleus, leading to transcription of

NCR-sensitive genes (

Beck and Hall, 1999; Bertram et al., 2000).

Null mutants of GLN3 or GAT1 in C. albicans were resistant to rap-

amycin, suggesting that the TOR signaling pathway acts through

Gln3 and Gat1 in this pathogen (

Liao et al., 2008).

The life cycle of the human pathogen Cryptococcus neoformans is

inﬂuenced by nitrogen availability, since in response to nitrogen

limitation this organism initiates monokaryotic fruiting or mating

(

Wickes et al., 1996). These two processes lead to the production of

spores, potential infectious propagules that are pathogenic in mice

(

Giles et al., 2009; Lengeler et al., 2000; Wickes et al., 1996). Fur-

thermore, the C. neoformans ammonium permease Amt2, which

is activated in response to nitrogen starvation, is required to induce

ammonium-responsive invasive growth and mating, indicating the

relevance of nitrogen metabolism in important aspects of C. neofor-

mans biology (

Rutherford et al., 2008). Here we report the identiﬁ-

cation of the GATA-type transcriptional activator Gat1 in C.

neoformans, which inﬂuences nitrogen uptake and controls the

transcription of genes involved in NCR, ergosterol biosynthesis,

iron uptake, cell wall organization and capsule biosynthesis.

2. Material and methods

2.1. Fungal strains, plasmids and media

C. neoformans H99 strain was the recipient for target gene dele-

tion. C. neoformans strains were maintained on YPD medium (1%

yeast extract, 2% peptone, 2% dextrose, and 1.5% agar). YPD plates

amended with hygromycin (200

g/ml) were used to select C. neo-

formans gat1 mutant strains. YPD plates amended with nourseo-

thricin (100

g/ml) were used to select C. neoformans gat1::GAT1

complemented strains. Plasmid pJAF15 (

Fraser et al., 2003) was

the source of a hygromycin resistance cassette and plasmid pAI4

(

Idnurm et al., 2004) was the source of a nourseothricin resistance

cassette.

2.2. In silico analysis of the C. neoformans GATA-type transcription

factor GAT1 ortholog

The putative C. neoformans GAT1 gene sequence was identiﬁed

by a BLAST search of the C. neoformans strain H99 genomic data-

base at the Broad Institute using GAT1 sequence of S. cerevisiae

[GenBank: NP_116632.1]. The amino acid sequences of Gat1 ortho-

logs from S. cerevisiae, C. albicans, A. nidulans, Ustilago maydis, Neu-

rospora crassa and C. neoformans were aligned using ClustalX2

(Larkin et al., 2007). Mega4 was utilized for phylogenetic analysis

applying the Neighbor-Joining method and the tree architecture

was inferred from 1000 bootstraps (

Tamura et al., 2007). Pfam

database (

http://pfam.sanger.ac.uk/) was used to search for con-

served domains in the Gat1 ortholog proteins.

2.3. Disruption and complementation of C. neoformans GAT1

The Delsgate methodology (

Garcia-Pedrajas et al., 2008) was

employed for disruption of GAT1. The hygromycin resistance cas-

sette from pJAF15 was subcloned into the EcoRV site of pDONR201

(Gateway donor vector, Invitrogen) to construct pDONRHYG plas-

mid. The 5

and 3

GAT1 ﬂanks (827 and 857 bp, respectively) were

PCR ampliﬁed, and gel puriﬁed using Illustra GFX PCR DNA and Gel

Band Puriﬁcation kit (GE Healthcare). Approximately 300 ng of

pDONRHYG vector and 30 ng of each PCR product were utilized

in the BP clonase reaction, according to manufacturer’s instruc-

tions (Invitrogen). This reaction was transformed into Escherichia

coli OmniMAX 2-T1. After conﬁrmation of the correct deletion

construct, the plasmid was linearized by I-SceI digestion prior to

C. neoformans biolistic transformation (

Toffaletti et al., 1993).

Transformants were screened by colony PCR, and the deletion

was conﬁrmed by Southern blot analysis and semi-quantitative

RT-PCR. For complementation, a 6.2 Kb genomic PCR fragment con-

taining the wild-type GAT1 gene was cloned into the SmaI site of

the pAI4 plasmid. The resulting plasmid was used for transforma-

tion of the gat1 mutant strain. Random genomic insertion of the

complemented gene was conﬁrmed by Southern blot analysis

and semi-quantitative RT-PCR. The primers utilized in these con-

structions are listed in

Table S1.

2.4. Phenotypic characterization assays

Nitrogen source utilization was assessed in YCB medium (Yeast

Carbon Base, Difco). Wild type (WT), gat1 mutant and comple-

mented strains were pre-cultured in YPD medium at 30 °C for

18 h. The cells were collected by centrifugation, washed three

times with sterile dH

O, suspended in YCB at a cell density of

cells/ml and incubated at 30 °C for 12 h to deplete nitrogen.

Cells suspensions (10

cells/ml) were serially 10-fold diluted and

l from each dilution was spotted onto YCB agar supplemented

with 2 mM of each amino acid, 100 mM of urea, or 37 mM of

ammonium sulfate. Sensitivity to rapamycin was assessed on

YPD agar medium supplemented with 100 ng/ml and 200 ng/ml

of rapamycin. WT, gat1 mutant and complemented cells were cul-

tured overnight in YPD, washed and suspended to a density of

cells/ml. The cells were diluted and spotted as described above.

As control, cells were grown in YPD agar only. The plates were

incubated for 2 days at 30 °C and photographed. Capsule formation

was examined by microscopy after incubation for 24 h at 30 °C in a

minimal medium and prepared with India ink. Relative capsule

sizes were deﬁned as the distance between the cell wall and the

capsule outer border by cell diameter. ImageJ software was utilized

to determine capsule measurements of one hundred cells of each

strain. The content of extracellular glucuronoxylomannan (GXM)

in culture supernatants was determined by ELISA (

Fonseca et al.,

2009

2.5. Thin Layer Chromatography

Analyses of amino acids in culture supernatants of WT and gat1

mutant strains were assessed by Thin Layer Chromatography (TLC).

Brieﬂy, starter cultures of WT and gat1 mutant cells were grown

overnight in YPD at 37 °C with shaking. Cells were washed three

times, suspended in YNB (with no ammonia nor amino acids and

amended with 2% of glucose) and incubated at 37 °C for 12 h to de-

plete nitrogen. Then, 10

cells of each strain were inoculated in

YNB supplemented with 2 mM proline or aspartic acid. After 20 h

incubation at 37 °C, the cells were removed by centrifugation for

10 min at 10,000g. The supernatants were passed through a

0.45

m pore ﬁlter, followed by an ultra-ﬁltration in membrane

disks (1 kDa pore size). Silica gel TLC plates were spotted with

l of each supernatant, and the mobile phase utilized for chroma-

tography was butanol: acetic acid: water (40:10:10). For amino

acids visualization, the plate was dipped in a ninhydrin solution

(0.2% in ethanol) for 10 s. After 30 min incubation at 60 °C, the

TLC plates were photographed. These analyses were performed in

biological and technical duplicates.

2.6. Macrophage infection assay

The susceptibility of fungal cells to the antifungal action of

phagocytes was determined by counting colony forming units

(CFU) after interaction of WT, gat1 mutant and gat1::GAT1 comple-

mented strains with the murine macrophage-like cell line RAW

264.7. Prior to interaction, fungal cells were opsonized with mono-

2 L. Kmetzsch et al. / Fungal Genetics and Biology xxx (2010) xxx–xxx

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

clonal antibody 18B7 (1

g/ml), a gift from Dr. Arturo Casadevall

(Albert Einstein College of Medicine, USA). Macrophages were

seeded at a concentration of 10

cells/well in a 96-well cell culture

plate, and incubated overnight at 37 °C in 5% CO

in Dulbecco’s

modiﬁed Eagle’s medium (DMEM) supplemented with 5% heat-

inactivated fetal bovine serum (FBS). Fungal cells (10

) were inoc-

ulated in each well, and after 1 h the wells were washed to remove

unattached, extracellular fungal cells. After 20 h of incubation, in-

fected cultures were again washed and sterile ice-cold dH

O was

added to each well to promote macrophage lysis. Fungal viability

was measured by plating the lysates on YPD for CFU determination

after cultivation of the plates for 48 h at 30 °C. The assay was per-

formed in triplicate sets for each strain. Student’s t test was used to

determine the statistical signiﬁcance of differences in fungal

survival.

2.7. Virulence assay

Virulence studies were conducted according to a previously de-

scribed intranasal inhalation infection model in mice (

Cox et al.,

2000). Fungal cells were cultured in 50 ml of YPD medium at

30 °C overnight with shaking, washed twice and re-suspended in

PBS. Groups of eight female BALB/c mice (approximately 5 weeks

old) were infected with 10

yeast cells suspended in 50

l PBS

and monitored daily. Kaplan–Meier analysis of survival was per-

formed using GraphPad Prism Software. Animal studies were ap-

proved by the Federal University of Rio Grande do Sul Ethics

Committee.

2.8. Microarray analysis

For RNA extraction, starter cultures of WT and gat1 mutant cells

were grown overnight in YPD at 37 °C with shaking. Cells were

washed three times and suspended in YNB with no ammonia nor

amino acids and amended with 2% of glucose. This medium was

supplemented with 10 mM proline as a nitrogen source and incu-

bated for 3 h at 37 °C. Three independent sets of RNA samples from

independent experiments were prepared using Trizol reagent

(Invitrogen) according to the manufacturer’s protocol. After DNase

treatment, RNA preparations were puriﬁed using RNAeasy mini

columns (Qiagen). The CyScribe First-Strand cDNA Labeling Kit

(GE Life Sciences) was utilized for preparation and puriﬁcation of

Cy3-dUTP and Cy5-dUTP labeled cDNA probes during ﬁrst-strand

cDNA synthesis reactions, according to the manufacture’s protocol.

These ﬂuorescent labeled cDNAs were synthesized from 25

g to-

tal RNA from each strain tested. Cy5-labeled cDNA from WT was

mixed with Cy3-labeled cDNA from the gat1 mutant strain and

the mixture was hybridized to C. neoformans microarray slides

for 18 h at 42 °C in a humid hybridization chamber. Arrays were

washed with SSC buffer (1Â) with 0.2% (w/v) SDS for 10 min at

room temperature, followed by two washes in SSC (0.1Â) with

0.2% (w/v) SDS for 5 min and scanned on a GenePix 4000B Scanner

(Molecular Devices). Image preprocessing and data quantiﬁcation

was done using GenePix Pro Software and the raw expression data

was obtained. For further data analysis, the TIGR microarray soft-

ware suite (

http://www.tm4.org/), which includes Midas and

MeV software, was used. Data sets were ﬁrst processed by Midas

using total-intensity and LOWESS normalizations, and standard-

deviation regulation, so that for each gene a normalized expression

value was attributed. Following, MeV Software was used to per-

form one sample Student’s t test in order to identify genes with

statistically signiﬁcant changes. Fold changes (WT/gat1

) were de-

rived from the mean expression levels from three independent

experiments. The complete list of differentially expressed genes

(2-fold up or down regulated) with P values of <0.05 is presented

Table S2. The C. neoformans arrays (version 2) were purchased

from an academic consortium of Genome Sequencing Center at

School of Medicine in Washington University in St. Louis (

http://

genomeold.wustl.edu/activity/ma/cneoformans/

2.9. Quantitative real time RT-PCR analysis

Real-time PCR reactions were performed in an Applied Biosys-

tems 7500 Real-Time PCR System. PCR thermal cycling conditions

were an initial step at 95 °C for 5 min followed by 40 cycles at 95 °C

for 15 s, 60 °C for 20 s and 72 °C for 20 s. Platinum SYBR green qPCR

Supermix (Invitrogen) was used as reaction mix, supplemented

with 5 pmol of each primer and 2

l of the cDNA template in a ﬁnal

volume of 25

l. All experiments were done in three independent

cultures and each cDNA sample was analyzed in duplicate with

each primer pair. Melting curve analysis was performed at the

end of the reaction to conﬁrm a single PCR product. Data was nor-

malized to actin cDNAs ampliﬁed in each set of PCR experiments.

Relative expression was determined by the 2

method (Livak

and Schmittgen, 2001

). The primers utilized in these experiments

are listed in

Table S1.

3. Results

3.1. Identiﬁcation of a GATA-type transcription factor Gat1 ortholog in

C. neoformans

The GAT1 gene [Broad Institute: CNAG_00193.2] was identiﬁed

in the C. neoformans var. grubii H99 genomic database available at

the Broad Institute (

http://www.broadinstitute.org/annotation/

genome/cryptococcus_neoformans/MultiHome.html

), based on its

similarity to GAT1 from S. cerevisiae. The C. neoformans GAT1 coding

region is 4239 bp long, contains three introns and encodes a puta-

tive 1277-amino-acid protein. Members of the GATA-factor family

of transcription factors have a GATA zinc-ﬁnger domain [Pfam:

PF00320] (

Marzluf, 1997) that is also present in the C. neoformans

Gat1 ortholog. Furthermore, a phylogenetic analysis including Gat1

sequences from different fungi species was performed (

Fig. 1A) and

showed that C. neoformans Gat1 is most similar to Gat1 from U.

maydis and least similar to Gat1 from S. cerevisiae. The domain

architecture of the C. neoformans Gat1 was compared to the ortho-

logs herein analyzed showing that all orthologs have the GATA

zinc-ﬁnger domain [Pfam: PF00320] plus a domain of unknown

function DUF1752 [Pfam: 08550] (

Fig. 1B).

3.2. The gat1 mutant exhibited normal growth only when arginine and

proline were used as sole nitrogen sources

In order to perform a functional analysis of Gat1 in C. neofor-

mans, knockout and complemented strains were constructed. Dele-

tion and complementation of GAT1 were conﬁrmed by Southern

blot analysis and semi-quantitative RT-PCR, as shown in

Fig. 2.

To assess the role of C. neoformans GAT1 in nitrogen uptake and

metabolism, the ability of WT, gat1 mutant and gat1::GAT1 com-

plemented strains to grow in distinct nitrogen sources was evalu-

ated. WT and complemented strains grew well on all nitrogen

sources tested, but slightly growth differences between these

strains were observed. However, the gat1 mutant had strongly im-

paired growth on isoleucine, leucine, alanine, asparagine, lysine,

aspartic acid, methionine, cysteine, phenylalanine, glutamic acid,

threonine, glutamine, tryptophan, glycine, valine, serine, tyrosine,

histidine, urea or ammonium sulfate as nitrogen sources. The

gat1 mutant exhibited normal growth only when the nitrogen

sources utilized were arginine and proline (

Fig. 3A). These gat1 mu-

tant phenotypes can be due to a nitrogen metabolism defect or to

an amino acid transport defect. To address this point, TLC analyses

L. Kmetzsch et al. / Fungal Genetics and Biology xxx (2010) xxx–xxx

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

of the supernatants of WT and gat1 mutant cultures in the presence

of proline or aspartic acid were performed. As seen in

Fig. 3B, after

20 h of incubation, proline was completely utilized by both WT and

gat1 mutant strains, in contrast with aspartic acid, which was con-

sumed only by WT strain. Since the aspartic acid concentration was

not altered in gat1 mutant supernatants in comparison to the con-

centration observed in the fresh medium, we can conclude that

gat1 mutant has a transport defect of some amino acids.

To test whether the TOR signaling pathway acts through the

activity of GAT1 in C. neoformans, as demonstrated for the orthologs

in C. albicans and S. cerevisiae (

Beck and Hall, 1999; Cardenas et al.,

1999; Liao et al., 2008

), we conducted rapamycin sensitivity as-

says. Unlike C. albicans and S. cerevisiae, the C. neoformans gat1 mu-

tant strain had almost identical sensibility to rapamycin in

comparison to WT and complemented strains (

Fig. 3C), indicating

that the loss of GAT1 does not inﬂuence the TOR signaling pathway

in C. neoformans.

3.3. Disruption of GAT1 does not inﬂuence capsule size, but decreases

extracellular GXM secretion

The gat1 mutant strain was tested for its ability to grow at 37 °C,

for capsule size and for melanin production since these traits are

considered key virulence factors in C. neoformans (

McClelland

et al., 2005). The loss of GAT1 apparently did not interfere with

any of these traits (

Fig. 4A and data not shown). The disruption of

GAT1, however, decreased the levels of extracellular GXM, since

lower polysaccharide contents were observed in culture superna-

tants from the gat1 mutant strain, in comparison to WT (P < 0.05)

(

Fig. 4B). This ﬁnding suggests a role for C. neoformans Gat1 on

the release of capsular polysaccharides to the extracellular

environment.

3.4. Gat1 is neither required for C. neoformans survival during

macrophage infection nor for virulence in a mouse intranasal model of

cryptococcosis

The GATA-type transcriptional regulators Gat1 of C. albicans and

AreA of A. fumigatus act in virulence of these human pathogens

(

Hensel et al., 1998; Limjindaporn et al., 2003). Therefore we tested

whether C. neoformans Gat1 is essential during macrophage infec-

tion in vitro and in mice. Unlike as occurs in C. albicans and

fumigatus, Gat1 does not inﬂuence C. neoformans virulence in mice

(

Fig. 4C). Furthermore, no differences were observed in survival of

WT, gat1 mutant and complement cells during an in vitro macro-

phage infection, as seen by CFU count showed in

Fig. 4D.

3.5. The GATA-type transcriptional activator Gat1 regulates genes

involved in NCR, ergosterol biosynthesis, iron uptake, cell wall

organization and capsule biosynthesis in C. neoformans

Microarray analyses were conducted to identify target genes

that are regulated by Gat1. We examined changes in global gene

expression due to deletion of GAT1 in the presence of proline, a

Fig. 1. Identiﬁcation of a GATA-type transcriptional activator Gat1 ortholog in C. neoformans A. Phylogenetic analysis applying the Neighbor-Joining method including Gat1

sequences from distinct fungi, as follows: S. cerevisiae [Genbank: NP_116632.1], C. albicans [Genbank: AAP50501.1], A. nidulans [Genbank: XP_681936.1], U. maydis [Broad

Institute: UM_04252], N. crassa [Genbank: AAB03891.1] and C. neoformans [Broad Institute: CNAG_00193.2]. The bar marker indicates the genetic distance, which is

proportional to the number of amino acid substitutions. B. Domain architecture in the Gat1 orthologs of different fungi. The GATA zinc-ﬁnger domain [Pfam: PF00320] is

represented by white ovals, and the domain of unknown function DUF1752 [Pfam: 08550] is represented by black bars. The length of each protein sequence (in amino acids)

is indicated in the right.

Fig. 2. Construction of C. neoformans GAT1 knockout and complemented strains. A. Gat1 deletion scheme. TV represents the targeting vector constructed by Delsgate

methodology. 5 GAT and 3 GAT represent the 5

and 3

gene ﬂanks of GAT1 gene, respectively. 5F and 5R: primers utilized to amplify 5

ﬂank of GAT1 gene. 3F and 3R: primers

utilized to amplify 3

ﬂank of GAT1 gene. Hyg

: cassette that confers hygromicin resistance. WT represents the wild type locus of the GAT1 gene in H99 strain.

represents the

GAT1 locus in the gat1 mutant strain. The cleavage sites of HindIII restriction enzyme are indicated. B. Conﬁrmation by Southern blot. Genomic DNA (10

g) from WT (lane 1),

gat1 mutant (lane 2) and gat1::GAT1 complemented (lane 3) strains were digested with HindIII restriction enzyme. The 3

gene ﬂank was used as probe in Southern

hybridization. Left numbers (in base pairs) indicate the hybridization signal sizes based upon the position of molecular size marker. C. Semi-quantitative RT-PCR using cDNA

from WT (lane 1), gat1 mutant (lane 2) and gat1::GAT1 complemented (lane 3) strains as template. Right numbers (in bp) indicate the length of the transcript ampliﬁcation for

GAT1 (upper panel) and ACT1 (lower panel) genes. Lane 4: positive control using genomic DNA as template. NC: negative control of the PCR reaction.

4 L. Kmetzsch et al. / Fungal Genetics and Biology xxx (2010) xxx–xxx

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

poor and non-repressing nitrogen source. We found that 127 genes

were differentially expressed at least 2-fold in the WT strain.

Among these, 54 genes were upregulated in WT in comparison to

gat1 mutant, including genes related to nitrogen metabolism and

NCR mechanism (proline dehydrogenase, glutamate dehydroge-

nase, and amino acid transporter), iron uptake (high-afﬁnity iron

permease FTR1), and ergosterol biosynthesis (squalene monooxy-

genase). Seventy-three genes were found to be downregulated in

the WT strain, including genes associated with capsule biosynthe-

sis (capsular associated proteins), carbohydrate metabolism

(alpha-

-rhamnosidase, UDP-glucose dehydrogenase, UDP-glucu-

ronic acid decarboxylase, alpha 1–3 mannosyltransferase), cell wall

integrity (chitin synthase 2), oxidative metabolism (copper zinc

superoxide dismutase), and signal transduction (protein kinase

C).

Table 1 summarizes the microarray results. Some genes with

less than 2-fold change were included in

Table 1 due to their rele-

vance to possibly explain the distinct phenotypes observed in the

gat1 mutant strain as that involved in capsule biosynthesis (capsu-

lar associated protein and alpha-1,3-mannosyltransferase) and

nitrogen metabolism (glutamate-rich WD repeat containing one

protein and amino acid transporter). The complete list of differen-

tially expressed genes (2-fold up and down regulated) with

P values of <0.05 is in Table S2. To validate the microarray results,

the differential expression of seven selected genes was conﬁrmed

by quantitative real time RT-PCR (

Fig. 5).

4. Discussion

Fungi can utilize a wide range of nitrogen sources and the key

regulators that control nitrogen acquisition are well conserved in

these organisms (

Marzluf, 1997). In the present study, the GATA-

type transcriptional activator Gat1 of C. neoformans was described.

GATA-factors are responsible for activation of NCR-sensitive genes,

as seen for Gat1 orthologs from S. cerevisiae, C. albicans and A.

fumigatus (

Hensel et al., 1998; Limjindaporn et al., 2003; Stanb-

rough et al., 1995

). Members of the GATA-factor family of tran-

scription factors are characterized by the presence of a GATA

zinc-ﬁnger domain [Pfam: PF00320] (

Marzluf, 1997). As expected,

C. neoformans Gat1 is required for optimal growth on a variety of

nitrogen sources, indicating that this GATA-factor is involved in

nitrogen uptake and metabolism. A similar response was observed

for C. albicans Gat1 and for Gln3, another GATA-type transcription

factor that acts in parallel to Gat1 to control nitrogen uptake in this

pathogen (

Liao et al., 2008; Limjindaporn et al., 2003). Further-

more, N. crassa nit-2 mutants had diminished growth on a diversity

of amino acids sources including branched chain amino acids, tryp-

tophan and phenylalanine, but not tyrosine (

Facklam and Marzluf,

1978).

The TOR signaling pathway, that regulates cell proliferation in

response to nutrient availability, is the target of inhibition by the

drug rapamycin in C. neoformans and C. albicans (

Cruz et al.,

2001). Rapamycin binds to FKBP12 protein, and this complex inter-

acts and inhibits the Tor1 kinase (

Cruz et al., 2001). The sensitivity

of C. albicans to rapamycin was greatly decreased by deletion of

either GLN3 or GAT1 (

Liao et al., 2008). However, C. neoformans

gat1 mutant strain had almost identical sensitivity to rapamycin

in comparison to WT and complemented strains, indicating that

the TOR signaling pathway does not act through Gat1 in C. neofor-

mans. This phenotype may indicate the presence of redundant

pathways or overlapping functions with other proteins that could

Fig. 3. Gat1 is involved in nitrogen uptake in C. neoformans. A. Nitrogen source utilization assay. Ten-fold serial dilutions of wild-type H99 (WT), gat1 mutant (gat1

) and

gat1::GAT1 complemented (gat1

::GAT1) cells were plated in YCB medium supplemented with different nitrogen sources, as indicated. B. Thin Layer Chromatography.

Culture supernatants of wild-type H99 (WT) and gat1 mutant strains were analyzed for amino acids (proline or aspartic acid) visualization. As controls, 2 mM of each amino

acid (C1) and fresh media supplemented with 2 mM of each amino acid (C2) were also spotted in the TLC plates. C. Rapamycin sensitivity assay. Ten-fold serial dilutions of

wild-type H99 (WT), gat1 mutant (gat1

) and gat1 ::GAT1 complemented (gat1

::GAT1) cells were plated in YPD medium supplemented with 200 ng/ml of rapamycin. The

plates were incubated for 2 days at 30 °C. As control, cells were grown in YPD agar only.

L. Kmetzsch et al. / Fungal Genetics and Biology xxx (2010) xxx–xxx

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

compensate the loss of GAT1 in C. neoformans during rapamycin

treatment.

The deletion of C. neoformans GAT1 did not affect capsule size,

but decreased the levels of extracellular GXM in culture superna-

Fig. 4. Evaluation of virulence-related phenotypes of the gat1 mutant strain. A. Relative capsule size of WT and gat1 mutant cells. B. Content of extracellular GXM in culture

supernatants of WT and gat1 mutant cells determined by ELISA.

P < 0.001. C. Virulence assay of WT, gat1 mutant and gat1::GAT1 complemented strains in an intranasal

inhalation infection model using BALB/c mice. D. Macrophage infection assay. CFU counting after macrophage infection with WT, gat1 mutant and gat1::GAT1 complemented

strains.

Table 1

List of GAT1-regulated genes in C. neoformans.

Category Description

/accession number at Broad Institute

or Genbank

ÃÃ

databases Fold change

(WT/gat1

)

Capsule biosynthesis Capsular associated protein|CNK01140

ÃÃ

0.54

Capsular associated protein|CNAG_00721

0.63

Carbohydrate metabolism Alpha-

-rhamnosidase|CNAG_02587

0.50

UDP-glucose dehydrogenase|CNAG_04969

0.22

UDP-glucuronic acid decarboxylase|CNG02560

0.35

Alpha-1,3-mannosyltransferase|CNAG_05142

1.54

Cell wall integrity Chitin synthase 2|CNAG_03326

0.56

Ergosterol biosynthesis Squalene monooxygenase|CNAG_06829

2.02

Squalene monooxygenase, putative|CND06110

2.01

Iron metabolism High-afﬁnity iron permease CaFTR1|CNAG_02959

2.45

Nitrogen metabolism Proline dehydrogenase|CNAG_02049

5.10

Glutamate dehydrogenase (NADP+)|CNC00920

ÃÃ

11.37

Glutamate-rich WD repeat containing 1|CNAG_01600

1.55

Amino acid transporter|CNAG_02539

1.42

Oxidative metabolism Copper zinc superoxide dismutase|CND01490

ÃÃ

0.22

Signal transduction Protein kinase C, putative|CNC03300

ÃÃ

0.42

Transporters Potassium transport protein|CNAG_02856

0.33

ATP-binding cassette (ABC) transporter|CND00300

ÃÃ

0.39

Low-afﬁnity zinc ion transporter, putative|CND00350

ÃÃ

0.45

Descriptions were obtained from NCBI database (http://www.ncbi.nlm.nih.gov/) and Broad Institute Database (http://www.broadinstitute.org/annotation/genome/

cryptococcus_neoformans/MultiHome.html

Data are presented as the average changes in expression of genes in WT cells compared with their expression in gat1 mutant cells during growth in YNB supplemented

with 10 mM proline as secondary nitrogen source. List contains a set of statistically signiﬁcant genes identiﬁed by Student’s t test that have average changes of greater than 2-

fold. Genes with less than 2-fold change (italicized) are included in

Table 1 due to their relevance to explain the distinct phenotypes observed in the gat1 mutant strain.

6 L. Kmetzsch et al. / Fungal Genetics and Biology xxx (2010) xxx–xxx

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

tants. Although the concentration of extracellular GXM is fre-

quently associated with capsule enlargement, this same phenotype

was observed for a sec6 mutant of C. neoformans (Panepinto et al.,

2009). We speculate that the basal levels of GXM secretion mani-

fested by the gat1 mutant are sufﬁcient to warrant capsule assem-

bly, although they are below the regular levels of polysaccharide

secretion by C. neoformans. The facts that Gat1 orthologs of C. albi-

cans and A. fumigatus are involved in virulence (

Hensel et al., 1998;

Limjindaporn et al., 2003

), together with our observation of de-

creased GXM levels in C. neoformans, led us to test the role of

Gat1 during macrophage infection in vitro and in a mice model of

cryptococcosis. No differences between WT, gat1 mutant and com-

plemented strains were observed in these assays, indicating that C.

neoformans Gat1 is not necessary for survival during host infection.

We hypothesize that this observation may be related to the fact

that arginine and proline are probably available in host tissues

and ﬂuids in their free form or obtained from the peptidase-med-

iated hydrolysis of host proteins (

Pinti et al., 2007). For example,

the utilization of serum amino acids by C. albicans gat1 mutant

and WT strains was examined. After 24 h of incubation, approxi-

mately 200

M of proline (corresponding to 100%) had been con-

sumed by both WT and gat1 mutant strains, which indicate the

availability and utilization of this amino acid by C. albicans in ser-

um (Limjindaporn et al., 2003). Another possibility is that nitrogen

transport in the C. neoformans gat1 mutant in the in vivo environ-

ment differs from that in the in vitro environment, which could

be sufﬁcient for virulence and survival in macrophages. However,

it remains unknown whether the observed phenotype for the

gat1 mutant in vitro also occurs during infection. Although the

expression of GAT1 was not required for fungal pathogenesis, we

cannot rule out the possibility that its product and related proteins

are required for infection, since nitrogen metabolism is relevant in

essential aspects of C. neoformans biology (

Kingsbury and McCus-

ker, 2008; Kingsbury et al., 2004; Wickes et al., 1996

C. neoformans transcription factor Gat1 activates the expression

of known NCR-sensitive genes, as occurs with GATA-factors ortho-

logs in other fungi. We found that genes encoding proline dehydro-

genase, glutamate dehydrogenase, and amino acid transporter, the

corresponding orthologs of S. cerevisiae PUT1, GDH2 and GAP1,

respectively, are upregulated in the WT strain in comparison with

gat1 mutant during growth with proline as nitrogen source. The

upregulation of such genes is in agreement with the observed ef-

fect on S. cerevisiae NCR-sensitive genes (

Hofman-Bang, 1999;

Scherens et al., 2006

). C. albicans Gat1 also regulates the expression

of the general amino acid transporter GAP1 ortholog (

Limjindaporn

et al., 2003

The expression of genes related to capsule biosynthesis and car-

bohydrate metabolism are also controlled by C. neoformans Gat1

(Table 1). One of the genes encoding capsular associated protein

is downregulated in the WT strain. Interestingly, UDP-glucose

dehydrogenase and UDP-glucuronic acid decarboxylase genes are

also downregulated. These enzymes directly participate in GXM

biosynthesis, which may explain the reduced levels of extracellular

GXM produced by the gat1 mutant. The basic building units of

GXM are UDP-glucuronic acid, UDP-xylose and GDP-mannose,

and the connections between these units are done by glycosyl-

transferases (reviewed in

Zaragoza et al. (2009)). Mannose is the

most abundant sugar unit in GXM followed by glucuronic acid.

The addition of glucuronic acid to the polysaccharide chain re-

quires UDP-glucuronic acid synthesis, which occurs via oxidation

of UDP-glucose by UDP-glucose dehydrogenase in the cytoplasm.

Xylose is the third component of GXM. UDP-xylose, the target of

fungal glycosyltransferases, is synthesized from the decarboxyl-

ation of UDP-glucuronic acid by UDP-glucuronic acid decarboxyl-

ase (UGD1) (reviewed in

Zaragoza et al. (2009)). C. neoformans

UGD1 is essential for growth at 37 °C and for capsule biosynthesis

(

Moyrand and Janbon, 2004). The gene encoding a

-1,3-manno-

syltransferase, which catalyzes the transfer of mannose from

GDP-mannose to

-1,3-linked mannose disaccharides (Sommer

et al., 2003), is upregulated in the WT in comparison to gat1 mu-

tant strain. Cryptococcal mannosyltransferase-1 gene (CMT1) was

shown to be homologous to C. neoformans CAP59, a gene involved

in capsule synthesis and GXM export (

Garcia-Rivera et al., 2004;

Zaragoza et al., 2009

). The loss of C. neoformans GAT1 negatively

inﬂuences the contents of released GXM in culture supernatants.

This phenotype, found in gat1 mutant strain, can be related to

the remodeled pattern of gene expression of key genes that act

in the GXM biosynthesis. The expression of squalene monooxygen-

ase, a rate-limiting step in sterol biosynthesis, is also reduced in

the gat1 mutant. A decrease in sterol production could potentially

impair vesicle biogenesis and secretion through unconventional

pathways, a mechanism used by C. neoformans to transport GXM

to extracellular environments (

Rodrigues et al., 2007).

The Sre1 transcription factor of C. neoformans regulates oxygen

sensing, sterol homeostasis and virulence (

Chang et al., 2007).

Gene expression analysis of WT and sre1 mutant strains in the

presence of cobalt chloride (a hypoxia mimicking agent) revealed

that genes involved in ergosterol biosynthesis and in iron/copper

transport are Sre1-regulated (

Lee et al., 2007). Surprisingly, at least

23 genes related to nitrogen metabolism were also regulated by

Sre1 during growth with 0.6 mM CoCl

, including C. neoformans

transcription factor GAT1 (transcriptional activator) [GenBank:

CNA01820] (Lee et al., 2007). As seen for Sre1, Gat1 also regulates

genes involved in ergosterol biosynthesis, cell wall integrity and

iron uptake. These ﬁndings suggest that these two transcription

factors are somehow linked and can act in parallel regulating

important pathways in C. neoformans.

5. Conclusions

In conclusion, we have shown that Gat1, a GATA-type transcrip-

tion factor in C. neoformans, is directly involved in nitrogen uptake

Fig. 5. Validation of the microarray results by quantitative real time RT-PCR.

Relative expression of amino acid transporter, proline dehydrogenase, high-afﬁnity

iron permease FTR1, alpha-1,3-mannosyltransferase, squalene monooxygenase,

chitin synthase 2, and capsular associated protein transcripts during growth of WT

and gat1 mutant cells in YNB amended with 10 mM of proline as nitrogen source.

The measured quantity of the mRNA in each of the samples was normalized using

the Ct values obtained for the actin gene. Data are shown as mean ± SD.

P < 0.05.

ÃÃ

P < 0.01.

L. Kmetzsch et al. / Fungal Genetics and Biology xxx (2010) xxx–xxx

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

and metabolism. Rapamycin sensitivity assays revealed that TOR

signaling pathway does not act through Gat1 in C. neoformans.

Additionally, Gat1 is neither required for C. neoformans survival

during macrophage infection nor for virulence in a mouse intrana-

sal model of cryptococcosis. As revealed by microarray analysis,

Gat1 controls the transcription of NCR-sensitive genes, and genes

involved in ergosterol biosynthesis, iron uptake, cell wall organiza-

tion and capsule biosynthesis in C. neoformans.

Acknowledgments

This work was supported by grants from the Brazilian agencies

Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico

(CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de

Nível Superior (CAPES, Brazil), Fundação de Amparo a Pesquisa

no Estado do Rio de Janeiro (FAPERJ, Brazil) and Financiadora de

Estudos e Projetos (FINEP, Brazil). The authors thank Dr. Joseph

Heitman and Dr. Alex Idnurm for providing pJAF15, pAI4 plasmids

and Dr. Arturo Casadevall for providing the monoclonal antibody

anti-GXM (18B7). Automated DNA sequencing was performed at

the facilities of the Brazilian Genome Network at the Center of Bio-

technology, CBiot-UFRGS-RS.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in

the online version, at

doi:10.1016/j.fgb.2010.07.011.

References

Beck, T., Hall, M.N., 1999. The TOR signalling pathway controls nuclear localization

of nutrient-regulated transcription factors. Nature 402, 689–692.

Bertram, P.G., Choi, J.H., Carvalho, J., Ai, W., Zeng, C., Chan, T.F., Zheng, X.F., 2000.

Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases. J. Biol. Chem.

275, 35727–35733.

Cardenas, M.E., Cutler, N.S., Lorenz, M.C., Di Como, C.J., Heitman, J., 1999. The TOR

signaling cascade regulates gene expression in response to nutrients. Genes

Dev. 13, 3271–3279.

Chang, Y.C., Bien, C.M., Lee, H., Espenshade, P.J., Kwon-Chung, K.J., 2007. Sre1p, a

regulator of oxygen sensing and sterol homeostasis, is required for virulence in

Cryptococcus neoformans. Mol. Microbiol. 64, 614–629.

Coffman, J.A., Cooper, T.G., 1997. Nitrogen GATA factors participate in

transcriptional regulation of vacuolar protease genes in Saccharomyces

cerevisiae. J. Bacteriol. 179, 5609–5613.

Coffman, J.A., Rai, R., Cunningham, T., Svetlov, V., Cooper, T.G., 1996. Gat1p, a GATA

family protein whose production is sensitive to nitrogen catabolite repression,

participates in transcriptional activation of nitrogen-catabolic genes in

Saccharomyces cerevisiae. Mol. Cell Biol. 16, 847–858.

Cox, G.M., Mukherjee, J., Cole, G.T., Casadevall, A., Perfect, J.R., 2000. Urease as a

virulence factor in experimental cryptococcosis. Infect. Immun. 68, 443–448.

Cruz, M.C., Goldstein, A.L., Blankenship, J., Del Poeta, M., Perfect, J.R., McCusker, J.H.,

Bennani, Y.L., Cardenas, M.E., Heitman, J., 2001. Rapamycin and less

immunosuppressive analogs are toxic to Candida albicans and Cryptococcus

neoformans via FKBP12-dependent inhibition of TOR. Antimicrob. Agents

Chemother. 45, 3162–3170.

Facklam, T.J., Marzluf, G.A., 1978. Nitrogen regulation of amino acid catabolism in

Neurospora crassa. Biochem. Genet. 16, 343–354.

Fonseca, F.L., Frases, S., Casadevall, A., Fischman-Gompertz, O., Nimrichter, L.,

Rodrigues, M.L., 2009. Structural and functional properties of the Trichosporon

asahii glucuronoxylomannan. Fungal Genet. Biol. 46, 496–505.

Fraser, J.A., Subaran, R.L., Nichols, C.B., Heitman, J., 2003. Recapitulation of the

sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. Gattii:

implications for an outbreak on Vancouver Island, Canada. Eukaryot. Cell. 2,

1036–1045.

Garcia-Pedrajas, M.D., Nadal, M., Kapa, L.B., Perlin, M.H., Andrews, D.L., Gold, S.E.,

2008. DelsGate, a robust and rapid gene deletion construction method. Fungal

Genet. Biol. 45, 379–388.

Garcia-Rivera, J., Chang, Y.C., Kwon-Chung, K.J., Casadevall, A., 2004. Cryptococcus

neoformans CAP59 (or Cap59p) is involved in the extracellular trafﬁcking of

capsular glucuronoxylomannan. Eukaryot. Cell. 3, 385–392.

Giles, S.S., Dagenais, T.R., Botts, M.R., Keller, N.P., Hull, C.M., 2009. Elucidating the

pathogenesis of spores from the human fungal pathogen Cryptococcus

neoformans. Infect. Immun. 77, 3491–3500.

Hardwick, J.S., Kuruvilla, F.G., Tong, J.K., Shamji, A.F., Schreiber, S.L., 1999.

Rapamycin-modulated transcription deﬁnes the subset of nutrient-sensitive

signaling pathways directly controlled by the Tor proteins. Proc. Natl. Acad. Sci.

USA 96, 14866–14870.

Hensel, M., Arst Jr., H.N., Aufauvre-Brown, A., Holden, D.W., 1998. The role of the

Aspergillus fumigatus areA gene in invasive pulmonary aspergillosis. Mol. Gen.

Genet. 258, 553–557.

Hofman-Bang, J., 1999. Nitrogen catabolite repression in Saccharomyces cerevisiae.

Mol. Biotechnol. 12, 35–73.

Idnurm, A., Reedy, J.L., Nussbaum, J.C., Heitman, J., 2004. Cryptococcus neoformans

virulence gene discovery through insertional mutagenesis. Eukaryot. Cell. 3,

420–429.

Jiang, Y., Broach, J.R., 1999. Tor proteins and protein phosphatase 2A reciprocally

regulate Tap42 in controlling cell growth in yeast. EMBO J. 18, 2782–2792.

Kingsbury, J.M., McCusker, J.H., 2008. Threonine biosynthetic genes are essential in

Cryptococcus neoformans. Microbiology 154, 2767–2775.

Kingsbury, J.M., Yang, Z., Ganous, T.M., Cox, G.M., McCusker, J.H., 2004. Cryptococcus

neoformans Ilv2p confers resistance to sulfometuron methyl and is required for

survival at 37 °C and in vivo. Microbiology 150, 1547–1558.

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam,

H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J.,

Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23,

2947–2948.

Lee, H., Bien, C.M., Hughes, A.L., Espenshade, P.J., Kwon-Chung, K.J., Chang, Y.C.,

2007. Cobalt chloride, a hypoxia-mimicking agent, targets sterol synthesis in

the pathogenic fungus Cryptococcus neoformans. Mol. Microbiol. 65, 1018–1033.

Lengeler, K.B., Wang, P., Cox, G.M., Perfect, J.R., Heitman, J., 2000. Identiﬁcation of

the MATa mating-type locus of Cryptococcus neoformans

reveals a serotype A

MATa strain thought to have been extinct. Proc. Natl. Acad. Sci. USA 97, 14455–

14460.

Liao, W.L., Ramon, A.M., Fonzi, W.A., 2008. GLN3 encodes a global regulator of

nitrogen metabolism and virulence of C. Albicans. Fungal Genet. Biol. 45, 514–

526.

Limjindaporn, T., Khalaf, R.A., Fonzi, W.A., 2003. Nitrogen metabolism and virulence

of Candida albicans require the GATA-type transcriptional activator encoded by

GAT1. Mol. Microbiol. 50, 993–1004.

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using

real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25,

402–408.

Magasanik, B., Kaiser, C.A., 2002. Nitrogen regulation in Saccharomyces cerevisiae.

Gene 290, 1–18.

Marzluf, G.A., 1997. Genetic regulation of nitrogen metabolism in the fungi.

Microbiol. Mol. Biol. Rev. 61, 17–32.

McClelland, E.E., Bernhardt, P., Casadevall, A., 2005. Coping with multiple virulence

factors: which is most important? PLoS Pathog. 1, e40.

Moyrand, F., Janbon, G., 2004. UGD1, encoding the Cryptococcus neoformans UDP-

glucose dehydrogenase, is essential for growth at 37 °C and for capsule

biosynthesis. Eukaryot. Cell. 3, 1601–1608.

Panepinto, J., Komperda, K., Frases, S., Park, Y.D., Djordjevic, J.T., Casadevall, A.,

Williamson, P.R., 2009. Sec6-dependent sorting of fungal extracellular

exosomes and laccase of Cryptococcus neoformans. Mol. Microbiol. 71, 1165–

1176.

Pinti, M., Orsi, C.F., Gibellini, L., Esposito, R., Cossarizza, A., Blasi, E., Peppoloni, S.,

Mussini, C., 2007. Identiﬁcation and characterization of an aspartyl protease

from Cryptococcus neoformans. FEBS Lett. 581, 3882–3886.

Rodrigues, M.L., Nimrichter, L., Oliveira, D.L., Frases, S., Miranda, K., Zaragoza, O.,

Alvarez, M., Nakouzi, A., Feldmesser, M., Casadevall, A., 2007. Vesicular

polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to

the problem of fungal trans-cell wall transport. Eukaryot. Cell. 6, 48–59.

Rutherford, J.C., Lin, X., Nielsen, K., Heitman, J., 2008. Amt2 permease is required to

induce ammonium-responsive invasive growth and mating in Cryptococcus

neoformans. Eukaryot. Cell. 7, 237–246.

Scherens, B., Feller, A., Vierendeels, F., Messenguy, F., Dubois, E., 2006. Identiﬁcation

of direct and indirect targets of the Gln3 and Gat1 activators by transcriptional

proﬁling in response to nitrogen availability in the short and long term. FEMS

Yeast Res. 6, 777–791.

Sommer, U., Liu, H., Doering, T.L., 2003. An alpha-1,3-mannosyltransferase of

Cryptococcus neoformans. J. Biol. Chem. 278, 47724–47730.

Stanbrough, M., Rowen, D.W., Magasanik, B., 1995. Role of the GATA factors Gln3p

and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated

genes. Proc. Natl. Acad. Sci. USA 92, 9450–9454.

Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary

genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599.

Thomas, G., Hall, M.N., 1997. TOR signalling and control of cell growth. Curr. Opin.

Cell Biol. 9, 782–787.

Toffaletti, D.L., Rude, T.H., Johnston, S.A., Durack, D.T., Perfect, J.R., 1993. Gene

transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J.

Bacteriol. 175, 1405–1411.

Wickes, B.L., Mayorga, M.E., Edman, U., Edman, J.C., 1996. Dimorphism and haploid

fruiting in Cryptococcus neoformans: association with the alpha-mating type.

Proc. Natl. Acad. Sci. USA 93, 7327–7331.

Zaragoza, O., Rodrigues, M.L., De Jesus, M., Frases, S., Dadachova, E., Casadevall, A.,

2009. The capsule of the fungal pathogen Cryptococcus neoformans. Adv. Appl.

Microbiol. 68, 133–216.

8 L. Kmetzsch et al. / Fungal Genetics and Biology xxx (2010) xxx–xxx

Please cite this article in press as: Kmetzsch, L., et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the hu-

man pathogen Cryptococcus neoformans. Fungal Genet. Biol. (2010), doi:

10.1016/j.fgb.2010.07.011

CURRICULUM VITAE

Nome: Débora Leite de Oliveira

Nascimento: 03/10/1983

Naturalidade: Rio de Janeiro

 Formação Acadêmica

 Bacharel em Microbiologia e Imunologia pela Universidade Federal do Rio de Janeiro, de

março de 2002 à dezembro de 2005.

 Doutorado em Química Biológica no Instituto de Bioquímica Médica – Universidade

Federal do Rio de Janeiro.

 Orientação de Monografia

 “O papel das proteínas Sec4 e Grasp na formação de vesículas extracelulares em

Saccharomyces cerevisiae”

Luna Sobrino Joffe, Monografia apresentada ao Instituto de Microbiologia –

Universidade Federal do Rio de Janeiro

 Atividade imunobiológica de vesículas secretadas pelo patógeno fúngico Candida

albicans

Gabriele Vargas Cesar, Monografia apresentada ao Instituto de Microbiologia –

Universidade Federal do Rio de Janeiro

 Comunicações em congress

 13 comunicações em congressos nacionais

 Publicações

1. Oliveira, Débora L. et al. Biogenesis of extracellular vesicles in yeast Many questions with few

answers, v. 3, p. 1-4, 2010.

2. OLIVEIRA, D. L. ; et al. Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage

Functions. Infection and Immunity, v. 78, p. 1601-1609, 2010

3. Oliveira, Débora L. et al. Characterization of Yeast Extracellular Vesicles: Evidence for the

Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis. Plos One , v. 5, p.

e11113, 2010.

4. Kmetzsch, Lívia ; et al. The GATA-type transcriptional activator Gat1 regulates nitrogen uptake

and metabolism in the human pathogen Cryptococcus neoformans. Fungal Genetics and Biology

(Print) , p. 1-2, 2010.

5. Kmetzsch, Lívia ; et al. The Vacuolar Ca2+ Exchanger Vcx1 is involved in Calcineurin-Dependent

Ca2+ Tolerance and Virulence in Cryptococcus neoformans.. Eukaryotic Cell , v. 10, p. 12, 2010.

Oliveira, et al. Biogenesis of extracellular vesicles in yeast Many questions with few answers, v. 3, p.

1-4, 2010.

6. Oliveira, Débora L. et al. Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation

reveals an intimate association between membrane lipids and glucuronoxylomannan. Fungal

Genetics and Biology, v. 46, p. 956-963, 2009.

7. RODRIGUES, M. L. ; et al. Extracellular vesicles produced by Cryptococcus neoformans contain

protein components associated with virulence. Eukaryotic Cell , v. 7, p. 58-67, 2008

8. RODRIGUES, M. L. et al. Vesicular Trans-Cell Wall Transport in Fungi: A Mechanism for the Delivery of

Virulence-Associated Macromolecules?. Lipid Insights , v. 2, p. 27-40, 2008.

9. RODRIGUES, M. L. et al. Vesicular Polysaccharide Export in Cryptococcus neoformans Is a

Eukaryotic Solution to the Problem of Fungal Trans-Cell Wall Transport (M.L.R., L.N., and D.L.O.

contributed equally to this work.). Eukaryotic Cell , v. 6, p. 48-59, 2007.

Livros Grátis
( http://www.livrosgratis.com.br )
 
Milhares de Livros para Download:
 
Baixar livros de Administração
Baixar livros de Agronomia
Baixar livros de Arquitetura
Baixar livros de Artes
Baixar livros de Astronomia
Baixar livros de Biologia Geral
Baixar livros de Ciência da Computação
Baixar livros de Ciência da Informação
Baixar livros de Ciência Política
Baixar livros de Ciências da Saúde
Baixar livros de Comunicação
Baixar livros do Conselho Nacional de Educação - CNE
Baixar livros de Defesa civil
Baixar livros de Direito
Baixar livros de Direitos humanos
Baixar livros de Economia
Baixar livros de Economia Doméstica
Baixar livros de Educação
Baixar livros de Educação - Trânsito
Baixar livros de Educação Física
Baixar livros de Engenharia Aeroespacial
Baixar livros de Farmácia
Baixar livros de Filosofia
Baixar livros de Física
Baixar livros de Geociências
Baixar livros de Geografia
Baixar livros de História
Baixar livros de Línguas

Baixar livros de Literatura
Baixar livros de Literatura de Cordel
Baixar livros de Literatura Infantil
Baixar livros de Matemática
Baixar livros de Medicina
Baixar livros de Medicina Veterinária
Baixar livros de Meio Ambiente
Baixar livros de Meteorologia
Baixar Monografias e TCC
Baixar livros Multidisciplinar
Baixar livros de Música
Baixar livros de Psicologia
Baixar livros de Química
Baixar livros de Saúde Coletiva
Baixar livros de Serviço Social
Baixar livros de Sociologia
Baixar livros de Teologia
Baixar livros de Trabalho
Baixar livros de Turismo
 
 