( PDF ) Estudo das proteínas da família gp82 das formas metacíclicas do Trypanosoma cruzi

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Vanessa Diniz Atayde

Estudo das proteínas da família gp82 das

formas metacíclicas do Trypanosoma cruzi

Tese apresentada à Universidade

Federal de São Paulo, Escola Paulista

de Medicina, para obtenção do Título

de Doutor em Ciências.

São Paulo

2008

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Vanessa Diniz Atayde

Estudo das proteínas da família gp82 das

formas metacíclicas do Trypanosoma cruzi

Orientadora: Dra. Nobuko Yoshida

Tese apresentada à Universidade

Federal de São Paulo, Escola Paulista

de Medicina, para obtenção do Título

de Doutor em Ciências.

São Paulo

2008

ads:

Atayde, Vanessa Diniz

Estudo das proteínas da família gp82 das formas metacíclicas do

Trypanosoma cruzi. / Vanessa Diniz Atayde. - - São Paulo, 2008.

xii, 77p.

Tese (Doutorado) – Universidade Federal de São Paulo. Escola

Paulista de Medicina. Programa de Pós-Graduação em Microbiologia,

Imunologia e Parasitologia.

Título em inglês: Study of gp82 family proteins of Trypanosoma

cruzi metacyclic forms.

1.Trypanosoma cruzi 2.Formas metacíclicas 3.Invasão celular

4.Clone CL-14 5.Proteínas da família gp82 6.Melanoma 7.Apoptose

Trabalho realizado na Disciplina de Parasitologia do Departamento

de Microbiologia, Imunologia e Parasitologia da Universidade

Federal de São Paulo, Escola Paulista de Medicina, com auxílios

financeiros concedidos pela Fundação de Amparo à Pesquisa do

Estado de São Paulo (FAPESP) e pelo Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq).

À Dra. Nobuko Yoshida, que me ensinou algo essencial

entre os “mil milhões” de coisas que me ensinou nesses

quase dez anos de convivência: a cientista que eu quero

me tornar um dia. (Sempre trabalhando intensamente!!!)

iii

Aos meus pais, Hely e Al, que SEMPRE me deram total

apoio e compreensão. À Dani, minha eterna irmãzinha.

NUNCA teria conseguido sem eles.

Agradecimentos

Foram quase dez anos na Disciplina de Parasitologia da UNIFESP. Aqui eu

obtive conhecimento, formação e exemplos que seguirei por toda a vida. Aqui eu

descobri que não há nada que eu realmente queira fazer além da ciência. Aqui eu fiz

amigos inesquecíveis que estarão sempre no meu coração.

Em primeiro lugar, agradeço à Dra. Nobuko Yoshida, por tudo que fez por mim

nesses anos de trabalho e aprendizado intensos. Tenho um enorme respeito e

admiração por ela, que é um dos meus maiores exemplos de vida. Sei que dificilmente

encontrarei alguém parecido daqui para frente, mas fico muito feliz por ter aprendido

tanto no tempo em que convivemos.

Agradeço a todos os Professores do Departamento, especialmente ao Dr.

Maurício Rodrigues e ao Dr. Renato Mortara, que foram muito importantes em

diferentes momentos da minha formação.

Agradeço à Ciça, minha melhor amiga, minha irmã, minha querida. Obrigada

pela paciência, pelas risadas, pelas longas conversas (e repetitivas...), e até mesmo

pelas broncas muitas vezes merecidas. Estou chegando amiga!

Agradeço à Fanny, minha grande (literalmente) e querida amiga. Suas histórias

malucas me fizeram rir até nos dias mais difíceis. Uma das melhores pessoas que

conheci na minha vida. Já estou com saudades.

Agradeço aos amigos do laboratório que contribuíram muito, cada um a seu

modo: Daniele, Renata S., Renata B., Marjorie, Fábio, Cláudio, Emanuelle, Carol,

Miriam, Simone, Érico, Carlinhos, Carla, Sílvia, Meire, Dú, Rafa, Silene, Gisa, Denis,

Josie, Paulo, Sheila, Jú, Mário....Espero encontrar todos vocês novamente!

Agradeço aos novos amigos, mas não menos importantes, Andrea Midory,

Fernando Maeda e Dani Staquicini. Aprendi muito com esses japinhas que têm um

grande caráter e um enorme coração. Tenho certeza que nossa amizade é e sempre

será 100%!

Agradeço aos amigos chilenos: Patrício Porcile, Sérgio Málaga, Ivan Neira,

Mauro Cortez (o Javi..., meu amigão!), Esteban Cordero e Charles Covarrubias.

Sentirei saudades da nossa convivência (principalmente das nossas festas...). Tenho

que agradecer especialmente ao Patricio Manque, meu primeiro contato com a

pesquisa. Obrigada pela sua amizade e por sempre acreditar na minha capacidade.

Agradeço à Mércia e à Regiane, sempre prestativas, amigas e muito elegantes!

E aos funcionários da disciplina: Dona Joaninha, Kleber, Luis, Lima, Adilson, Cidinha,

Dona Fátima e Seu Adalton. Um agradecimento especial à Sandra, minha fofa!

Por fim, agradeço à minha família muito unida e feliz, que tornou possível a

realização dessa tese. Com o apoio deles, tudo acabou ficando mais fácil. Amo vocês!

“Navegadores antigos tinham uma frase gloriosa: “Navegar

é preciso, viver não é preciso.”

Quero para mim o espírito desta frase, transformada a

forma para a casar com o que eu sou: Viver não é necessário; o

que é necessário é criar.

Não conto gozar a minha vida; nem em gozá-la penso. Só

quero torná-la grande, ainda que para isso tenha de ser o meu

corpo e a minha alma a lenha desse fogo.

Só quero torná-la de toda a humanidade; ainda que para

isso tenha de a perder como minha.

Cada vez mais assim penso. Cada vez mais ponho na

essência anímica do meu sangue o propósito impessoal de

engrandecer a pátria e contribuir para a evolução da humanidade.

É a forma que em mim tomou o misticismo da nossa Raça.”

Fernando Pessoa (Obra Poética)

vii

Índice

Resumo ix

Abstract xi

Introdução: Trypanosoma cruzi e Doença de Chagas

1. Doença de Chagas 1

2. Trypanosoma cruzi, agente etiológico da Doença de Chagas 4

3. Cepas e grupos do Trypanosoma cruzi 7

4. Infecção oral pelo Trypanosoma cruzi 10

5. Mecanismos de invasão celular pelas formas metacíclicas do

Trypanosoma cruzi 12

5.1. Gp82: glicoproteína de 82kDa expressa na superfície das

formas metacíclicas envolvida na invasão celular 13

5.2. Outras moléculas de superfície das formas metacíclicas

envolvidas na invasão celular 16

5.3. Vias de sinalização ativadas durante a invasão das formas

metacíclicas do Trypanosoma cruzi 20

Capítulo 1 – Bases moleculares da avirulência do clone CL-14 do

Trypanosoma cruzi

Artigo: “Molecular basis of non-virulence of Trypanosoma cruzi

clone CL-14” 24

viii

Capítulo 2 – Expressão e localização celular de moléculas da família

gp82 em formas metacíclicas do Trypanosoma cruzi

Artigo: “Expression and cellular localization of molecules of the

gp82 family in Trypanosoma cruzi metacyclic trypomastigotes” 28

Capítulo 3 – Indução de apoptose em células de melanoma pela gp82

em sua forma recombinante

Artigo: “A recombinant protein based on Trypanosoma cruzi surface

molecule gp82 induces apoptotic cell death in melanoma cells” 39

Discussão 50

Referências Bibliográficas 60

Resumo

Nosso principal objetivo foi caracterizar a família gp82 de proteínas,

codificada pela família gênica gp82, que é parte da grande família multigênica

gp85/trans-sialidase do T. cruzi. Até o início desse estudo, somente a gp82 de

superfície, envolvida na invasão celular das formas metacíclicas da cepa CL e

identificada pelo anticorpo monoclonal (MAb) 3F6, havia sido descrita.

Na primeira parte, investigamos as bases da avirulência in vivo e in vitro

das formas metacíclicas do clone CL-14 do T. cruzi, derivado da cepa CL.

Descobrimos que a baixa infectividade desses parasitas está associada com a

reduzida expressão da gp82 na superfície, reforçando o papel central da

molécula na infecção pelo T. cruzi. Além disso, os dados sugeriram que devido

à deficiência da gp82, as formas metacíclicas do clone CL-14, como as da cepa

G, utilizam preferencialmente as glicoproteínas gp35/50 para interagir com a

célula hospedeira.

Na segunda parte, analisamos a expressão e a localização de moléculas

da família gp82 em formas metacíclicas das cepas G e CL do T. cruzi. Para

isso, isolamos um novo membro (clone C03) que, em contraste com o clones

de cDNA representantes da família gp82 previamente descritos, J18 (cepa G) e

R31 (cepa CL), não contém o epítopo do MAb 3F6. Na análise comparativa da

seqüência de aminoácidos do clone C03, observamos 59,1% de identidade

com o clone J18 e 60,2% de identidade com o clone R31. Além disso, quando

alinhamos as seqüências de aminoácidos dos clones C03 e Tc-85 (gp85 das

formas tripomastigotas de cultura de tecido), observamos 57,2% de identidade,

indicando que esse clone também é parte da família gp85/trans-sialidase.

Utilizando os anticorpos policlonais anti-J18 e anti-C03, e o MAb 3F6,

mostramos que membros da família gp82 são expressos em formas

metacíclicas na superfície e intracelularmente, e que alguns desses membros

têm localização diferenciada em parasitas dos grupos T. cruzi I e II. Ao

contrário da gp82 reativa com o MAb 3F6, específica das formas metacíclicas,

outras moléculas da família foram detectadas também nas formas

tripomastigotas de cultura de tecido e amastigotas pelos anticorpos policlonais.

Na terceira parte, investigamos o efeito da gp82 em sua forma

recombinante (J18), sobre a linhagem de melanoma murino Tm5. Observamos

que J18 liga-se a essas células induzindo a despolimerização dos filamentos

de actina similar à citocalasina-D, droga indutora de apoptose em células de

mamíferos. Em vista disso, fizemos uma análise comparativa das alterações no

citoesqueleto de actina e eventos característicos de morte celular por apoptose

nas células Tm5 tratadas com J18, e na linhagem de melanócitos melan-a, da

qual Tm5 é derivada. Mostramos que J18 tem função indutora de apoptose

somente sobre as células do melanoma Tm5. Descrevemos assim uma

propriedade de um membro da família gp82, nunca antes descrita para nenhum

outro componente molecularmente definido do T. cruzi.

Abstract

The main goal of this work was to characterize the proteins encoded by

the gp82 gene family, which is part of the large T. cruzi gp85/trans-sialidase

multigenic family. Previous studies had focused on the surface gp82, which is

engaged in the cell invasion by CL strain metacyclic forms and is identified by

monoclonal antibody (MAb) 3F6.

Firstly, we investigated the molecular basis of the non-virulence of T.

cruzi clone CL-14 metacyclic forms in vivo and in vitro. We found that the low

infectivity of these parasites is associated with reduced expression of surface

gp82, reinforcing the role of this molecule in T. cruzi infection. In addition, our

data suggested that instead of gp82, metacyclic forms of clone CL-14, like G

strain, preferentially engage gp35/50 glycoproteins to interact with the host cell.

Secondly, we analyzed the expression and cellular localization of

molecules of the gp82 family in T. cruzi metacyclic forms of G and CL strains.

We cloned a new member of this family, designated C03, which lacks the MAb

3F6 epitope, in contrast to the previously described gp82 cDNA clones J18 (G

strain) and R31 (CL strain). The predicted amino acid sequence of the C03

clone displayed 59,1% identity to J18 clone and 60,2% to R31 clone. When the

amino acid sequences of C03 and Tc-85 (tissue culture trypomastigote gp85)

clones were aligned, the identity was 57,2%, indicating that this new clone

belongs to the gp85/trans-sialidase family. Using anti-J18 and anti-C03

polyclonal antibodies, as well as MAb 3F6, we demonstrated that members of

the gp82 family are localized on the cell surface and intracellularly in metacyclic

forms, and some members have different cellular localization in parasites from

T. cruzi I and II groups. As opposed to metacyclic stage-specific gp82 identified

by MAb 3F6, other gp82 family molecules were also detected in tissue culture

trypomastigotes and amastigotes by polyclonal antibodies.

Thirdly, we investigated the effect of gp82, as a recombinant protein

(J18), on the murine melanoma lineage Tm5. We observed that J18 binds to

xii

these cells disrupting actin filaments similarly to cytochalasin-D, drug that

induces apoptosis in mammalian cells. Based on these information, we

comparatively analyzed alterations in actin cytoskeleton and apoptotic events in

J18-treated Tm5 cells, and in the melanocyte lineage melan-a, from which Tm5

is derived. J18 selectively induced apoptosis in Tm5 melanoma cells. Our

results show an activity of a protein from gp82 family that has not been

described for any other T. cruzi molecule.

Introdução: Trypanosoma cruzi e Doença de Chagas

1. Doença de Chagas

A doença de Chagas ou tripanossomíase americana foi descoberta pelo

brasileiro Carlos Chagas (Fig.1) em 1909, às margens do Rio São Francisco

(MG), enquanto combatia uma epidemia de malária que atrasava a construção

da ferrovia Central do Brasil. Com a identificação do agente etiológico e a

descrição da moléstia, Chagas foi o primeiro cientista a apontar a importância

sócio-econômica da doença no país. Além disso, fundou as bases da pesquisa

básica realizada em todo o século XX até os dias atuais, essencial no

entendimento das interações parasita-hospedeiro.

Figura 1. Carlos Chagas (FIOCruz).

Eram os “anos de ouro” da medicina tropical, ciência prestigiada na

época em razão da necessidade de manter íntegra a saúde dos soldados

brancos e colonizadores do final do século XIX, assolados pelas doenças

tropicais. O Império Britânico, que se estendia a diversos países da África,

transformava a medicina tropical em negócio comercial e militar (Worboys,

1993). Nesse período emergiram os “caçadores de parasitas”, como Ross e

Laveran, que ganharam o prêmio Nobel em 1902 e 1907 por suas descobertas

em malária. Chagas foi nomeado em 1913 e 1921 sem ser premiado, pois a

comunidade científica não se convencia da importância e do impacto da sua

descoberta (Coutinho et al., 1999).

Atualmente, a doença de Chagas é considerada uma das doenças

tropicais negligenciadas causadas por protozoários, junto com a

tripanossomíase africana e a leishmaniose (Boutayeb et al., 2007). Segundo a

Organização Mundial de Saúde (WHO, 2008), as doenças tropicais

negligenciadas afetam mais de 1 bilhão de indivíduos em todo o mundo,

causando aproximadamente 500.000 mortes anuais. A maior parte dos

doentes, que vivem em situações de extrema pobreza, encontra-se co-

infectada com mais de um patógeno, já que na maioria dos países afetados

ocorre concomitantemente mais de uma doença (Reddy et al., 2007) (Fig.2). As

interações entre os patógenos que co-infectam um indivíduo podem ser

sinérgicas ou competitivas e, sendo assim, uma doença pode interferir no

impacto geral da outra (Buck et al., 1978, Petney & Andrews, 1998).

Figura 2. Ocorrência global das doenças tropicais negligenciadas. Cinza: uma

doença, azul: duas, verde: três, amarelo: quatro, laranja: cinco, vermelho: seis

(gnntdc.sabin.org).

Apesar da grande importância sócio-econômica, poucos esforços são

despendidos no combate às doenças tropicais negligenciadas, provavelmente

porque não afetam países ricos. Nos últimos 30 anos, somente 1% dos

investimentos mundiais no desenvolvimento de novas drogas foram

direcionados ao tratamento dessas doenças (Beyrer et al., 2007, Reddy et al.,

2007).

A doença de Chagas é um grave problema de saúde pública de países

da América Latina, sendo endêmica do norte do México ao sul da Argentina

(Fig.3). Segundo a WHO (2005), existem aproximadamente 13 milhões de

indivíduos infectados e cerca de 14.000 mortes anuais, causadas

principalmente pelos danos irreversíveis ao coração, característicos da doença.

Figura 3. Endemia global da doença de Chagas (Morel & Lazdins, 2003).

Populações rurais vivendo em habitações de baixa qualidade, onde o

inseto vetor se estabelece, são os principais alvos da doença de Chagas. Em

países do cone sul da América Latina como Argentina, Brasil e Chile, medidas

de controle da proliferação do inseto vetor têm sido eficientes na diminuição da

incidência anual, que passou de 700.000-800.000, estimativa de 1990, para

200.000 casos (WHO, 2005). Além disso, dos 100 milhões de indivíduos

vivendo sob risco de infecção em 1990, hoje existem apenas 40-75 milhões

(Schofield et al., 2006, Coura, 2007). Entretanto, essas medidas de controle

não são eficazes contra os insetos silvestres ou peridomiciliares, que podem

eventualmente reinfestar as casas ou contaminar os alimentos (Schofield et al.,

2006).

2. Trypanosoma cruzi, agente etiológico da Doença de Chagas

O Trypanosoma cruzi é um protozoário flagelado pertencente à ordem

Kinetoplastidae, que se caracteriza pela presença de cinetoplasto, única

mitocôndria que contém uma complexa rede de DNA em seu interior. É

digenético, pois necessita de um hospedeiro vertebrado e de um hospedeiro

invertebrado para completar seu ciclo de vida. Os hospedeiros invertebrados

são os insetos triatomíneos da família Reduviidae, conhecidos popularmente

como “barbeiros”. Os membros dessa família que têm maior importância

epidemiológica são o Triatoma infestans, Triatoma brasiliensis, Triatoma

dimidiata, Rhodnius prolixus e Panstrongylus megistus (Schofield, 2000).

Três formas do T. cruzi são identificadas durante seu ciclo de vida:

amastigotas e epimastigotas, replicativas, e tripomastigotas (sanguíneas ou

metacíclicas), infectivas (Fig.4).

Figura 4. Formas tripomastigota, amastigota e epimastigota (uta.edu).

A transmissão vetorial inicia-se quando o inseto vetor, após o repasto

sanguíneo, defeca liberando as formas tripomastigotas metacíclicas. Essas

formas penetram o hospedeiro vertebrado pela lesão da picada ou diretamente

pelas mucosas, onde invadem as células das camadas subjacentes. Dentro

das células, as formas metacíclicas encontram-se em um compartimento

derivado da membrana plasmática e de lisossomos, o vacúolo parasitóforo, de

onde escapam e diferenciam-se em amastigotas. As formas amastigotas

replicam-se no citoplasma e em seguida diferenciam-se em tripomastigotas,

liberados após a ruptura celular. Os tripomastigotas infectam células vizinhas

ou caem na corrente sanguínea, espalhando a infecção aos diferentes órgãos e

tecidos. As formas tripomastigotas sanguíneas circulantes são ingeridas pelo

barbeiro durante o repasto e diferenciam-se em formas epimastigotas, que se

replicam na porção média do seu intestino e migram à porção final, onde se

diferenciam em formas tripomastigotas metacíclicas, completando o ciclo

(Fig.5).

Figura 5. Ciclo de vida do T. cruzi (WHO).

Há três ciclos de transmissão vetorial do T. cruzi. O ciclo de maior

importância epidemiológica é o doméstico, já que perpetua a infecção nos

seres humanos. No ciclo silvestre, participam triatomíneos que, uma vez

contaminados, infectam roedores, marsupiais e outros animais silvestres. O

terceiro ciclo é o peridoméstico, do qual participam roedores, marsupiais, gatos

e cães que entram e saem das residências, e os insetos silvestres atraídos às

casas pela disponibilidade de alimento. Esse ciclo serve de ligação entre os

ciclos doméstico e silvestre (Rey, 2002). Um estudo recente mostrou que em

91,5% das vilas rurais da região oeste da Venezuela, onde a doença de

Chagas é endêmica, foram encontrados cães infectados com T. cruzi,

convivendo diretamente com humanos infectados em casas que continham

ninhos do inseto vetor. Isso mostra que animais domésticos são importantes

fatores de risco na transmissão da doença de Chagas aos humanos (Crisante

et al., 2006).

Além da transmissão vetorial, também ocorrem as transmissões

congênita, transfusional ou por transplante de órgãos. Esses tipos de

transmissão têm levado a doença de Chagas às regiões não-endêmicas, como

a América do Norte, através da migração de indivíduos infectados (Buekens et

al., 2008). A transmissão oral, que será discutida adiante, é de grande

importância na constante manutenção do ciclo silvestre e tornou-se evidente

devido aos casos de infecção humana por ingestão de alimentos contaminados

com o parasita.

A doença é caracterizada por uma fase aguda, com sintomas que

surgem alguns dias após a contaminação, e por uma fase crônica, com

sintomas que surgem após um período latente de 10 a 30 anos. A lesão inicial,

ou chagoma de inoculação, chama particularmente a atenção quando ocorre

no olho (sinal de Romaña) (Fig.6). Os sintomas da fase aguda são oriundos da

ruptura das células infectadas e da presença de parasitas no sangue, o que

desencadeia uma resposta inflamatória com febre, edema, linfadenopatia e

miocardite (Rey, 2002). Em 95% dos casos, essa fase é assintomática e

somente 5% dos indivíduos sintomáticos morrem. As lesões da fase crônica

afetam irreversivelmente órgãos como o coração, esôfago e intestino, pois uma

fibrose difusa ocupa o lugar das áreas inflamadas. De 30% dos pacientes

crônicos que desenvolvem manifestações clínicas, 95% têm o coração afetado.

Infecções pelo HIV podem reativar a doença em pacientes crônicos,

evidenciando a persistência do parasita no hospedeiro durante essa fase

(Punnukollu et al., 2007, Teixeira et al., 2008). No entanto, a maioria dos

infectados tem a forma indeterminada da doença de Chagas, sem nunca

desenvolver a doença crônica (Rey, 2002).

Figura 6. Sinal de Romaña (tmcr.usuhs.mil).

O tratamento, quando o doente tem acesso a ele, é insatisfatório devido

à sua ineficácia no estágio crônico, além dos diversos efeitos tóxicos. As

drogas utilizadas são o nifurtimox e o beznidazol, que têm efeitos significativos

na fase aguda da doença, com mais de 80% de cura parasitológica em

pacientes tratados (Rassi et al., 2000). Porém, sua eficácia varia em diferentes

regiões geográficas, provavelmente devido às variações de sensibilidade das

cepas do parasita aos medicamentos, conforme demonstrado

experimentalmente e no homem (Brener et al., 1976, Andrade et al., 1992).

3. Cepas e grupos do Trypanosoma cruzi

Os isolados ou cepas do T. cruzi, provenientes de vários hospedeiros

selvagens ou domésticos, vertebrados ou invertebrados, apresentam-se

altamente heterogêneos segundo critérios morfológicos, genéticos, bioquímicos

e clínicos (Devera et al., 2003). O elevado grau de diversidade do parasita

pode estar associado à sua necessidade de adaptação e sobrevivência em

diferentes hospedeiros.

Analisando-se padrões de isoenzimas, as cepas do T. cruzi foram

inicialmente divididas em três zimodemas, Z1, Z2 e Z3 (Miles et al., 1980). Em

seguida, Dvorak et al. (1982) mostraram que as cepas possuem diferentes

quantidades de DNA total por parasita, sugerindo que a heterogeneidade

biológica poderia ser resultante de diferenças genéticas. A partir desse

momento, o maior desafio foi identificar marcadores moleculares que se

correlacionassem com o comportamento variável das cepas estudadas.

Em 1996, Souto et al. agruparam os isolados do T. cruzi em duas

linhagens, através da análise da seqüência do rDNA 24S e das seqüências

intergênicas dos genes de mini-éxons. Amostras de diferentes hospedeiros de

vários países da América Latina foram classificadas em linhagem 1 (Z2) ou

linhagem 2 (Z1). Cepas pertencentes ao Z3 não puderam ser classificadas,

uma vez que apresentaram seqüências incomuns (Mendonça et al., 2002). Em

1999, num Simpósio Internacional, a comunidade científica sugeriu a

padronização da nomenclatura: o que foi originalmente classificado como

linhagem 1 e linhagem 2, foi renomeado como grupo T. cruzi II e grupo T. cruzi

I, respectivamente (Satellite-Meeting, 1999). Posteriormente, o grupo T. cruzi II

foi subdividido em cinco subgrupos (IIa-IIe), resultantes de fusões genéticas

com sinais de recombinação e heterozigose (Brisse et al., 2000, Westenberger

et al., 2005). Em outras análises, o Z3 foi caracterizado como parte de um

grupo ancestral chamado T. cruzi III (Pedroso et al., 2007). O clone CL-Brener

do projeto genoma (El-Sayed et al., 2005), que contém marcadores de DNA

dos grupos T. cruzi I, II e III sendo considerado um clone híbrido, foi agrupado

ao sub-grupo IIe (Elias et al., 2005, Pedroso et al., 2007, Cerqueira et al.,

2008).

Estabelecidos os critérios de classificação dos isolados, iniciaram-se os

estudos que tinham como objetivo associar os grupos genéticos com

características epidemiológicas e clínicas da doença de Chagas. Em muitos

estudos, a variabilidade nos sintomas foi atribuída à diversidade das cepas

(Baptista et al., 2006). Uma explicação para essa diversidade seria a expressão

diferencial de genes específicos. No caso das formas metacíclicas, diferentes

cepas apresentam repertórios de superfície distintos e interagem com as

células-alvo de modos particulares (Yoshida, 2006).

Em análise epidemiológica de cepas de animais silvestres e domésticos,

vetores e humanos de áreas endêmicas e não-endêmicas do Brasil, Colômbia

e Bolívia, observou-se a associação do grupo T. cruzi II com o ciclo doméstico

e do grupo T. cruzi I, composto principalmente por isolados de gambás, com o

ciclo silvestre. Esses grupos foram considerados filogeneticamente divergentes

(Briones et al., 1999, Zingales et al., 1999). Cepas pertencentes ao grupo T.

cruzi III, freqüentemente encontradas em triatomíneos silvestres, representam

um reservatório constitutivo de infecção humana na Amazônia (Pedroso et al.,

2007).

O grupo T. cruzi II possui alta incidência em doentes crônicos de regiões

endêmicas do estado de Minas Gerais (Lages-Silva et al., 2006). Esse grupo

também é freqüentemente identificado em pacientes crônicos da Argentina e

Chile, podendo ser o único que causa a doença nesses países (Di Noia et al.,

2002). Já o grupo T. cruzi I é endêmico no norte da América do Sul, incluindo a

Amazônia, onde a doença de Chagas é emergente devido às microepidemias

por infecção oral e aos doentes com sintomas severos (Coura et al., 2002,

Miles et al., 2003, Teixeira et al., 2006). Recentemente, uma cepa do grupo T.

cruzi I (cepa José-IMT) foi isolada de um paciente crônico com graves sintomas

cardíacos (Teixeira et al., 2006), mostrando que esse grupo também pode

causar a doença crônica. Esse paciente provinha de uma vila rural do estado

da Paraíba, próxima a Catolé do Rocha onde, em 1986, 26 pessoas se

contaminaram num surto de infecção oral após a ingestão de caldo de cana,

com um caso fatal (Shikanai-Yasuda et al., 1991).

4. Infecção oral pelo Trypanosoma cruzi

Análises filogenéticas sugerem que a infecção pelo T. cruzi iniciou-se há

milhões de anos em animais silvestres, da maneira que ainda persiste em

regiões de florestas selvagens como a Amazônia (Briones et al., 1999, Coura,

2007). Apesar de existirem relatos de infecção humana em múmias de até

9.000 anos (Aufderheide et al., 2004), a doença endêmica somente se

estabeleceu com o desmatamento das florestas, nos últimos 300 anos (Aragão,

1983). Com a colonização de áreas próximas ao homem pelos triatomíneos,

que se adaptaram ao novo nicho alimentando-se do sangue humano e de

animais domésticos, a infecção tornou-se uma zoonose, a doença de Chagas

(Coura, 2007).

No ciclo silvestre, a contaminação oral com o T. cruzi mantém constante

a infecção dos animais, devido ao hábito de se alimentarem de pequenos

mamíferos e insetos (Jansen et al., 1997). Os gambás (Fig.7) têm um

importante papel nesse ciclo, já que não somente carregam as formas

sanguíneas, mas também são reservatórios de formas metacíclicas,

encontradas no lúmen de sua glândula anal (Deane, 1984). Por outro lado,

animais domésticos podem invadir o ambiente selvagem para caçar,

infectarem-se por via oral e levarem a infecção para o ambiente domiciliar

(Coura, 2007).

Figura 7. Gambás (Didelphis marsupialis).

No caso do homem, com o controle da proliferação do inseto vetor e dos

bancos de sangue, a infecção oral é hoje a mais importante forma de

transmissão da doença de Chagas (Coura, 2006). Mais da metade dos casos

de doença aguda, registrados entre 1968 e 2000 na Amazônia brasileira, são

atribuídos a microepidemias causadas por infecção oral após a ingestão de

alimentos contaminados com o T. cruzi (Coura et al., 2002). Esse tipo de

infecção está em destaque desde 2005, quando foram relatados 26 casos no

estado de Santa Catarina, resultantes da ingestão de caldo de cana com o

parasita. Os focos da contaminação foram triatomíneos moídos ou somente

suas fezes contendo formas metacíclicas, especializadas na invasão do epitélio

da mucosa gástrica em camundongos (Hoft, 1996, Hoft et al., 1996). Outros

eventos semelhantes foram relatados nos estados do Amazonas, Amapá e

Pará (Coura et al., 2002, Promed-mail, 2007) (Fig.8). Os alimentos ingeridos

pelos seres humanos também podem ser contaminados pela secreção da

glândula anal de gambás. Em 1986, na Paraíba (Fig.8), onde diversas pessoas

adquiriram a doença aguda após ingerir caldo de cana, gambás altamente

infectados foram encontrados (Shikanai-Yasuda et al., 1991).

Figura 8. Microepidemias de doença de Chagas por infecção oral no Brasil.

5. Mecanismos de invasão celular pelas formas metacíclicas do

Trypanosoma cruzi

O T. cruzi invade diversos tipos celulares, incluindo células epiteliais,

musculares, macrófagos e fibroblastos, e esse é um fator essencial no

estabelecimento da infecção do hospedeiro vertebrado. Em ensaios in vitro,

inúmeros isolados do parasita e linhagens de células de mamíferos, têm sido

utilizados (Fig.9). Esses ensaios são imprescindíveis no estudo do processo de

invasão, onde interagem vários componentes do parasita e da célula

hospedeira, ativando vias de sinalização bidirecionais que promovem a

internalização (Burleigh & Andrews, 1998, Mortara et al., 2005, Yoshida, 2006).

É importante lembrar que as estratégias de invasão celular utilizadas pela

forma tripomastigota de cultura de tecido (referente à forma sanguínea) e pela

forma metacíclica são distintas. Apesar de ambas serem infectivas, cada uma

possui seu repertório de moléculas de superfície e interage de maneira

particular com as células-alvo.

Figura 9. Cepas ou clones do T. cruzi e células de mamíferos utilizados em

ensaios de invasão (adaptado de Yoshida, 2006).

Nas infecções naturais, as formas metacíclicas são as primeiras a

entrarem em contato com as células do hospedeiro. A capacidade infectiva

dessas formas difere entre as cepas, devido à expressão diferencial de

moléculas de superfície. Normalmente, cepas pertencentes ao grupo T. cruzi II

são mais infectivas in vitro do que cepas pertencentes ao grupo T. cruzi I (Ruiz

et al., 1998, Neira et al., 2002, Yoshida, 2006). In vivo, essa correlação nem

sempre existe, já que fatores do hospedeiro devem ser considerados

(Covarrubias et al., 2007).

Em estudos com formas metacíclicas de cepas muito infectivas, como

por exemplo a cepa CL (Fig.10), têm-se confirmado o papel central da molécula

de superfície gp82 na invasão celular, in vitro e in vivo (Ramirez et al., 1993,

Santori et al., 1996, Ruiz et al., 1998, Yoshida et al., 2000, Neira et al., 2003).

Figura 10. Forma metacíclica da cepa CL marcada com anticorpo anti-gp82.

5.1. Gp82: glicoproteína de 82kDa expressa na superfície das formas

metacíclicas envolvida na invasão celular

Responsável pela interação inicial das formas metacíclicas com a célula

hospedeira, a gp82 promove a invasão de cepas altamente infectivas como a

cepa CL (Ramirez et al., 1993, Ruiz et al., 1998). Essa glicoproteína contém

oligossacarídeos N-ligados (Ramirez et al., 1993) e está inserida na membrana

plasmática via âncora de GPI, forma de ancoramento de muitas proteínas

eucarióticas (Schenkman et al., 1988, Cardoso de Almeida & Heise, 1993). A

porção glicídica da gp82 não está envolvida na invasão, e o sítio de adesão

celular encontra-se no domínio central da molécula e é formado pela

justaposição de duas seqüências peptídicas carregadas separadas por outra

hidrofóbica (Santori et al., 1996, Manque et al., 2000).

As evidências da importância da gp82 no processo de invasão são: i) o

anticorpo monoclonal (MAb) 3F6, contra uma porção do domínio central da

gp82 adjacente ao sítio de adesão celular, inibe a entrada do parasita em

células não-fagocíticas (Ramirez et al., 1993); ii) tanto a gp82 nativa como a

sua forma recombinante inibem a penetração do parasita na célula hospedeira

(Ramirez et al., 1993, Santori et al., 1996); iii) a ligação da gp82 ao seu

receptor, ainda não identificado, leva à mobilização de Ca

intracelular em

ambas as células (Dorta et al, 1995, Ruiz et al., 1998), evento essencial para a

invasão (Yakubu et al., 1994, Moreno et al., 1994, Tardieux et al., 1994).

Formas epimastigotas não induzem sinal intracelular de Ca

(Tardieux et al.,

1994, Dorta et al., 1995), ao menos que estejam transfectadas com um vetor

de expressão carregando a seqüência de cDNA da gp82 (Manque et al., 2003).

Na infecção in vivo, a gp82 tem função central na invasão do epitélio da

mucosa gástrica, quando formas metacíclicas são inoculadas oralmente em

camundongos (Hoft, 1996, Hoft et al., 1996, Neira et al., 2003). A primeira

evidência do envolvimento da gp82 na infecção oral foi a inibição da

infectividade da cepa CL pelo tratamento com o MAb 3F6. Anticorpos contra

outras proteínas de superfície do T. cruzi não apresentaram o mesmo efeito.

Além disso, foi visto que a gp82 é capaz de ligar-se diretamente à mucina

gástrica, sendo possivelmente essa a primeira etapa no acesso das formas

metacíclicas às células da mucosa (Neira et al., 2003). No epitélio gástrico de

camundongos infectados oralmente, ninhos de amastigotas podem ser

observados (Fig.11). Uma particularidade relevante para a infecção oral é a

resistência da gp82 às condições do meio gástrico. Parasitas da cepa CL,

expostos ao suco gástrico in vivo ou tratados com pepsina in vitro se mantêm

infectivos às células de mamíferos, pois esses tratamentos não afetam a

integridade da gp82 (Neira et al., 2003, Cortez et al., 2006a).

As formas metacíclicas do T. cruzi também infectam a mucosa ocular.

Cães inoculados com essas formas desenvolvem altas parasitemias em

comparação com os inoculados com as formas sanguíneas, que causam

parasitemias muito baixas ou indetectáveis (Bahia et al., 2002). Se há função

da gp82 na invasão da mucosa ocular ainda precisa ser determinado.

Figura 11. Ninhos de amastigotas da cepa CL no epitélio da mucosa gástrica de

camundongo infectado por via oral. Corte histológico corado com hematoxilina-

eosina (Yoshida, 2008).

Posteriormente, o papel da gp82 no estabelecimento da infecção oral foi

confirmado através do estudo dos isolados gp82-deficientes, 569 e 588 (Cortez

et al., 2003). Esses isolados produzem baixas parasitemias em camundongos

quando comparados com a cepa CL. Porém, in vitro, apresentam

comportamento similar à cepa CL, pois expressam a gp30. Essa molécula de

superfície é também reconhecida pelo MAb 3F6 e tem atividade sinalizadora de

, mas não se liga à mucina gástrica eficientemente. Fica claro que a

invasão das células-alvo pelas formas metacíclicas pode ser promovida tanto

pela gp82 como pela gp30, que preserva o domínio de adesão celular da gp82.

Entretanto, a eficiente infecção da mucosa gástrica depende da gp82, devido à

sua capacidade de interagir com a mucina (Fig.12).

Figura 12. Interações entre as formas metacíclicas e a mucosa gástrica durante

infecção oral (adaptado de Yoshida, 2008).

5.2. Outras moléculas de superfície das formas metacíclicas envolvidas

na invasão celular

As moléculas gp35/50 e gp90 também estão envolvidas na invasão das

formas metacíclicas. Como a gp82, essas moléculas ligam-se à célula

hospedeira de maneira receptor-dependente (Ruiz et al., 1998), porém até o

momento, nenhum dos receptores foi descrito. Cepas pouco infectivas, como a

cepa G, parecem utilizar preferencialmente as gp35/50 para interagir com as

células (Yoshida et al., 1989). Essas moléculas não são efetivas como a gp82,

provavelmente devido à sua reduzida atividade sinalizadora de Ca

(Dorta et

al., 1995). Isolados pouco infectivos expressam também a gp90, que atua

como modulador negativo da invasão (Málaga & Yoshida, 2001).

Expressas na superfície de formas metacíclicas e de epimastigotas de

todos os isolados de T. cruzi analisados até o momento, as glicoproteínas

gp35/50 são moléculas do tipo mucina (Yoshida, 2006). O envolvimento dessas

moléculas na invasão celular foi determinado através da inibição da

infectividade de formas metacíclicas pelo MAb 10D8, direcionado a um epítopo

de carboidrato das gp35/50, ou pelas proteínas nativas purificadas (Yoshida et

al., 1989, Ruiz et al., 1993).

As glicoproteínas do tipo mucina são codificadas por uma família

multigênica que representa aproximadamente 1% do genoma do T. cruzi (Di

Noia et al., 1998, Buscaglia et al., 2006). São ancoradas à membrana via GPI e

contém em sua estrutura açúcares O-ligados a resíduos de treonina através de

uma N-acetilglicosamina, ao invés de uma N-acetilgalactosamina, como

normalmente é observado nas mucinas de vertebrados (Schenkman et al.,

1993, Acosta-Serrano et al., 2001). As moléculas de formas epimastigotas e

metacíclicas diferem entre si somente na porção lipídica da âncora GPI, tendo

a mesma porção glicídica (Acosta-Serrano et al., 1995). Dependendo da cepa

do T. cruzi, as mucinas contém resíduos de galactofuranose além dos resíduos

de galactopiranose (Previato et al., 1994, Acosta-Serrano et al., 1995, Acosta-

Serrano et al., 2001), apresentando um grande polimorfismo (Mortara et al.,

1992). O MAb 10D8, que reconhece as gp35/50 de isolados pouco invasivos

como as cepas G e Tulahuén, reage com epítopos de galactofuranose,

enquanto o MAb 2B10 reage com epítopos de galactopiranose, presentes em

todas as cepas (Yoshida, 2006).

Na transferência de moléculas de ácido siálico do hospedeiro pela

enzima trans-sialidase, as gp35/50 atuam como principais aceptores. Essa

enzima, aproximadamente 30 vezes mais ativa em tripomastigotas de cultura

de tecido (TCT) do que em formas metacíclicas, transfere resíduos de ácido

siálico às galactoses disponíveis, e age como uma sialidase (neuraminidase)

na ausência desses açúcares aceptores (Schenkman et al., 1991a, Schenkman

& Eichinger, 1993, Acosta-Serrano et al., 2001). Dependendo da cepa do T.

cruzi, os resíduos de ácido siálico presentes nas gp35/50 atrapalham a

invasão. Por exemplo, o tratamento da cepa G com neuraminidase bacteriana,

aumenta a infectividade desta cepa. Além disso, as gp35/50 dessialiladas têm

maior atividade sinalizadora de Ca

na célula hospedeira (Yoshida et al.,

1997). Em contraste, a infectividade da cepa CL não é afetada pelo tratamento

com neuraminidase (Yoshida et al., 1997), já que essa cepa utiliza a gp82 para

a sua internalização. Quanto a papel das moléculas gp35/50 na infecção oral

pelas formas metacíclicas, que são altamente resistentes à proteólise, elas

devem proteger os parasitas da digestão pelo suco gástrico (Mortara et al.,

1992).

Ao contrário da gp82 e das mucinas gp35/50, a glicoproteína gp90,

específica de formas metacíclicas, atua como regulador negativo da invasão

celular. A função inibitória da gp90 sobre a invasão foi demonstrada em

experimentos onde oligonucleotídeos anti-senso, complementares à porção C-

terminal da molécula onde aparentemente está o sítio de adesão celular, foram

utilizados no tratamento de formas metacíclicas da cepa G. O tratamento

reduziu a expressão da molécula e aumentou significativamente a infectividade

dos parasitas (Málaga e Yoshida, 2001). A gp90, presente em múltiplas cópias

no genoma do T. cruzi (Franco et al., 1993), contém oligossacarídeos N-ligados

e é ancorada à membrana via GPI (Schenkman et al., 1988, Yoshida et al.,

1990). Apesar de ligar-se à célula-alvo de maneira receptor-dependente, não

induz sinal intracelular de Ca

(Ruiz et al., 1998). A infectividade das diferentes

cepas tem correlação inversa com a expressão de gp90, como por exemplo a

cepa CL, que expressa baixos níveis da molécula (Ruiz et al., 1998). Estudos

recentes com dois isolados de pacientes, 573 e 587, poderiam explicar a

severidade de alguns casos de doença aguda após a ingestão de formas

metacíclicas. Os dois isolados são pouco infectivos in vitro, porém, in vivo, a

cepa 573 produz altas parasitemias, semelhante à cepa CL. Isso se dá pelo

fato da gp90 da cepa 573 ser suscetível à degradação pelo suco gástrico

(Cortez et al, 2006a).

A análise da expressão de moléculas de superfície pelas formas

metacíclicas tem se revelado um bom método para diferenciar cepas

pertencentes a grupos filogeneticamente divergentes, que diferem na

infectividade in vitro. Em análise de 10 cepas, de diferentes fontes e regiões

geográficas, observou-se que os parasitas altamente invasivos são deficientes

em gp90 e gp35/50 identificadas respectivamente pelos MAbs 1G7 e 10D8,

embora expressem formas variantes dessas moléculas, reconhecidas pelos

MAbs 5E7 e 2B10. Cepas pouco invasivas expressam gp90 reativa com os

MAbs 1G7 e 5E7, e gp35/50 reconhecidas pelos MAbs 2B10 e 10D8. A

expressão de gp82 reativa com o MAb 3F6 é ubíqua entre as cepas (Yoshida,

2006) (Fig.13).

Figura 13. Invasão celular e expressão na superfície de cepas do T. cruzi

(adaptado de Yoshida, 2006).

5.3. Vias de sinalização ativadas durante a invasão das formas

metacíclicas do Trypanosoma cruzi

A eficiência na invasão depende da molécula de superfície que é

utilizada pelo parasita para interagir com a célula hospedeira. Após essa

interação, distintas vias de sinalização são ativadas em ambas as células,

como observado em estudos com as cepas G e CL, pertencentes a grupos

genéticos divergentes (Yoshida, 2006, Yoshida e Cortez, 2008). Essas vias

foram identificadas através do tratamento dos parasitas ou das células-alvo

com drogas já descritas para células de mamíferos (Neira et al., 2002, Ferreira

et al., 2006).

Formas metacíclicas da cepa G utilizam preferencialmente as moléculas

gp35/50 para interagir com a célula hospedeira (Yoshida et al., 1989, Yoshida

et al., 1997). Após a ligação da gp35/50 ao seu receptor, é ativada no parasita

a via de sinalização que envolve a adenilato ciclase, gerando AMP cíclico

(cAMP) e liberação de Ca

de ácidocalciossomos (Neira et al., 2002), vacúolos

ácidos contendo um sistema de troca Ca

(Docampo et al., 1995) (Fig.14).

Por outro lado, a invasão celular pelas formas metacíclicas da cepa CL é

mediada pela gp82 (Ramirez et al., 1993). Após a ligação da gp82 ao seu

receptor, é ativada no parasita a via de sinalização que envolve a fosfolipase C

(PLC), com geração de trifosfato de inositol (IP

), e liberação de Ca

compartimentos IP

-dependentes (Yoshida et al., 2000, Neira et al., 2002). Em

amastigotas, epimastigotas ou TCT, a liberação de Ca

dependente de IP

não

é detectada (Moreno et al., 1994, Docampo et al., 1995). Além disso, após a

interação da gp82 com a célula hospedeira, é fosforilada no parasita uma

proteína de 175 kDa, de função desconhecida (Favoreto et al., 1998, Neira et

al., 2002) (Fig.14). Os tripanossomatídeos Leishmania, Trypanosoma brucei e

Trypanosoma cruzi possuem um grande repertório de proteínas quinases,

compreendendo aproximadamente 2% de seus genomas, sugerindo que a

fosforilação tem função chave na biologia desses parasitas. Entretanto, não

possuem proteínas tirosino-quinases típicas de eucariotos (El-Sayed et al.,

2005, Parsons et al., 2005).

Figura 14. Vias de sinalização ativadas nas formas metacíclicas das cepas G e

CL durante a invasão.

Na célula-hospedeira, duas vias de sinalização distintas são ativadas

durante a invasão de formas metacíclicas das cepas G ou CL (Cortez et al.,

2006b, Ferreira et al., 2006). A via ativada pela cepa G envolve o cAMP,

enquanto que, na via ativada pela cepa CL, estão envolvidas a PI3-quinase

(PI3-K) e a proteína quinase C (PKC), com liberação de Ca

intracelular IP

dependente (Ferreira et al., 2006). Muitos componentes das vias ativadas,

tanto no parasita como na célula hospedeira, ainda precisam ser estudados.

Muitos patógenos intracelulares subvertem vias de sinalização da célula

hospedeira em seu próprio benefício, como por exemplo, as bactérias Shigella

e Escherichia coli enteroinvasiva, que induzem o remodelamento do

citoesqueleto celular durante a invasão (Bourdet-Sicard et al., 2000, Cossart &

Sansonetti, 2004). Ferreira et al. (2006) observaram que a invasão de formas

metacíclicas das cepas G ou CL é dependente ou independente do

citoesqueleto de actina da célula hospedeira, respectivamente. Esse estudo

esclareceu parte das discrepâncias em relação ao papel do remodelamento da

actina na invasão pelo T. cruzi, ao mostrar que a dependência ou não da actina

pode ser determinada pela molécula de superfície que é utilizada pelo parasita

na invasão. Após a interação da molécula com seu receptor na célula-alvo,

diferentes vias de sinalização levando à reorganização da actina são ativadas.

Em estudos anteriores, Schenkman e Mortara (1992) observaram que a

entrada das formas metacíclicas não é afetada pelo tratamento das células

com citocalasina D, enquanto Osuna et al. (1993) observaram que o processo

de invasão dessas formas é significativamente inibido por latrunculina B, droga

que desorganiza o citoesqueleto de actina. Resultados contrastantes também

foram encontrados em experimentos com formas TCT. Alguns estudos

mostraram que a internalização dessas formas é independente do

citoesqueleto de actina da célula hospedeira, pois o tratamento das células

com citocalasina D, droga que despolimeriza a F-actina, não inibe ou aumenta

a invasão dos parasitas (Schenkman et al., 1991b, Tardieux et al., 1994).

Outros estudos mostraram que a citocalasina D tem efeito inibitório na invasão

de diversos tipos celulares, incluindo fibroblastos, mioblastos, cardiomiócitos e

macrófagos (Barbosa & Meirelles, 1995, Rosestolato et al., 2002).

Objetivos do Estudo

Capítulo 1 – Bases moleculares da avirulência do clone CL-14 do

Trypanosoma cruzi

Investigar as causas da avirulência das formas metacíclicas do clone

CL-14, em comparação com a cepa CL, com foco no papel da gp82 de

superfície.

Capítulo 2 – Expressão e localização celular de moléculas da família gp82

em formas metacíclicas do Trypanosoma cruzi

Caracterizar o repertório das moléculas gp82 expressas nas formas

metacíclicas através da análise da expressão e localização celular nas duas

cepas filogeneticamente distintas, G e CL.

Capítulo 3 – Indução de apoptose em células de melanoma pela gp82 em

sua forma recombinante

Investigar o efeito pró-apoptótico da proteína gp82 do T. cruzi, em sua

forma recombinante (J18), sobre as células do melanoma Tm5 e sobre a

linhagem de melanócitos melan-a.

Capítulo 1

Bases moleculares da avirulência do clone

CL-14 do Trypanosoma cruzi

Artigo:

“Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14”

Atayde, V., Neira, I., Cortez, M., Ferreira, D., Freymüller, E., Yoshida, N.

International J. Parasitology, 2004, 34: 851-860

As formas metacíclicas da cepa CL (Brener & Chiari, 1963), que utilizam

a gp82 de superfície na invasão celular, são altamente infectivas in vitro e in

vivo (Ruiz et al., 1998, Neira et al., 2003). Em contraste, as formas metacíclicas

do clone CL-14, derivado da cepa CL (Chiari, 1981), não produzem infecção

patente mesmo quando inoculadas em camundongos recém–nascidos (Lima et

al., 1990). No entanto, elicitam mecanismos imunológicos protetores contra

desafios letais com formas sanguíneas de cepas virulentas (Lima et al., 1990,

Paiva et al., 1999). É possível que a manutenção do clone CL-14 em culturas

axênicas por longos períodos tenha contribuído para a sua atenuação (Lima et

al., 1990).

Nosso principal objetivo foi determinar os fatores associados com a

baixa infectividade do clone CL-14. A invasão celular in vitro com formas

metacíclicas desse isolado é possível e foi utilizada em nosso estudo, sempre

em comparação com a cepa parental, CL (Fig.15). Os resultados mostraram

que a reduzida capacidade de invasão do clone CL-14 está associada com a

baixa expressão da gp82 em sua superfície e que, aparentemente, utiliza a

gp35/50 para a sua internalização. Esses dados reforçam a importância da

gp82 da superfície das formas metacíclicas no processo de invasão celular.

CL-14

Figura 15. Invasão celular in vitro por formas metacíclicas da cepa CL e do clone

CL-14. Notar a reduzida quantidade de parasitas do clone CL-14 (Giemsa, 1000x).

Experimentos complementares:

Imagens de células HeLa contendo amastigotas da cepa CL e do clone

CL-14 do T. cruzi

A figura 1C do artigo mostra que o clone CL-14 não é capaz de se

replicar intracelularmente. Em ensaios mantidos por 72 horas após a invasão

com as formas metacíclicas, quase não foram encontrados amastigotas

intracelulares do clone CL-14. No caso da cepa CL, muitas células foram

encontradas contendo um grande número de amastigotas. Na figura 16,

apresentamos imagens que ilustram essa observação.

Figura 16. Replicação intracelular da cepa CL e do clone CL-14 (Giemsa, 1000x).

Níveis “steady-state” dos transcritos gp82 da cepa CL e do clone CL-14

do T. cruzi

Os níveis “steady-state” dos transcritos gp82 de formas metacíclicas da

cepa CL e do clone CL-14 foram analisados por “northern blot”. O RNA total

extraído dos parasitas foi hibridizado com a sonda J18, produzida a partir de

um fragmento de cDNA que contém a fase de leitura aberta do gene gp82,

mesma sonda utilizada para as análises de “southern blot” do artigo. Em

ambos, foi revelado um fragmento de aproximadamente 2.4 Kb (Fig.17). Esse

resultado corrobora os dados do artigo que mostram que o clone CL-14, apesar

de não expressar a gp82 na superfície, contém a molécula intracelular.

Figura 17. Níveis “steady-state” dos transcritos gp82 da cepa CL e do clone CL-

14 do T. cruzi. (A) Formas metacíclicas (1 x 10

) foram ressuspendidas em 1 ml de

Trizol (Invitrogen). Após a homogeneização, foram adicionados 200 l de clorofórmio e

a amostra foi centrifugada a 10.800 rpm, 15 min. À fase aquosa, foram adicionados

500 l de isopropanol. Em seguida, a amostra foi centrifugada, lavada com etanol

70% e depois de seca, ressuspendida em água com dietilpirocarbonato a 0.05%.

Quantidades de RNA (~8 g) extraído foram desnaturadas a 70

C por 15 min em

tampão de amostra (50 l de MOPS 10X (MOPS 41.6g/l, acetato de sódio 6.76g/l,

EDTA 2.72g/l, pH 7.0), 90 l de formaldeído 37%, 250 l de formamida e 2 l de

solução de azul de bromofenol 0.01% em MOPS 1X). Após a desnaturação, as

amostras foram mantidas em gelo e momentos antes da aplicação no gel (1%

agarose, 10% formaldeído em MOPS 1X) foram adicionados 1.5 l de brometo de

etídio. (B) Para análise por “northern blot”, após a eletroforese, o RNA do gel foi

transferido à membrana e em seguida ligado covalentemente ao nylon através da

exposição à luz UV (150 mJ), no sistema GS Gene Linker UV Chamber (Bio-Rad). Em

seguida, a membrana foi hibridizada com a sonda J18. Sonda α-tubulina foi utilizada

como controle positivo do experimento. Após a hibridização, a membrana foi exposta

em filme de raio-X por 72 horas a -70

C. Os números à esquerda indicam tamanhos

moleculares.

Capítulo 2

Expressão e localização celular de moléculas

da família gp82 em formas metacíclicas

do Trypanosoma cruzi

Artigo:

“Expression and cellular localization of molecules of the gp82 family

in Trypanosoma cruzi metacyclic trypomastigotes”

Atayde, V., Cortez, M., Souza, R., Franco da Silveira, J., Yoshida, N.

Infection and Immunity, 2007, 75: 3264-3270

Pelo menos 50% do genoma do T. cruzi é composto por seqüências

repetidas, que podem ser retrotransposons, repetições subteloméricas ou

seqüências pertencentes às famílias que codificam proteínas de superfície.

Essas famílias multigênicas (MASP, trans-sialidases, mucinas e proteases

gp63), representam aproximadamente 18% dos 22.570 genes codificadores de

proteínas do parasita (El-Sayed et al., 2005).

O T. cruzi apresenta em seu genoma aproximadamente 60 cópias de

seqüências homólogas ao gene gp82, formando a família gênica gp82 (Araya

et al., 1994). Essa família é parte da grande família gp85/trans-sialidase,

composta por 1430 genes, representando aproximadamente 6% dos genes

codificadores do parasita (El-Sayed et al., 2005). Através da análise da

estrutura e expressão de genes da família gp82, Songthamwat et al. (2007)

identificaram três subfamílias gênicas gp82, com expressão de RNA

mensageiro (mRNA) estágio-específica nas formas metacíclicas.

A gp82 apresenta 53.3% de identidade de aminoácidos com a gp85,

molécula envolvida na invasão das formas TCT que contém sítios de ligação à

laminina e à citoqueratina 18 (Giordano et al., 1999, Magdesian et al., 2001).

As regiões de homologia incluem os motivos Asp (SXDXGXTW), descritos nas

neuraminidases bacterianas, e o motivo VTV (VTVXNVFLYNR), específico do

T. cruzi, ambos característicos da família gp85/trans-sialidase (Crennell et al.,

1993, Cross & Takle, 1993, Frasch, 2000) (Fig.18). Apenas 25% da família

conservam o motivo VTV, provavelmente devido à forte pressão seletiva sobre

seus membros, que são alvos do sistema imune tanto por respostas humorais

como celulares (El-Sayed et al., 2005, Tzelepis et al., 2008). No extremo final

das duas seqüências há uma região hidrofóbica que é sinal para adição de

âncora GPI (Araya et al., 1994, Giordano et al., 1999) (Fig.18).

Duas moléculas gp82 de superfície, reativas com o MAb 3F6, foram

caracterizadas a partir de clones de cDNA das cepas G (J18) e CL (R31). J18 e

R31 possuem identidade de 97,9% entre suas seqüências peptídicas (Araya et

al., 1994, Ruiz, 1998, Yoshida et al., 2006).

Figura 18. Alinhamento das seqüências de aminoácidos da gp85 (clone Tc85-11,

GeneBank AAD13347.1) e da gp82 (clone J18, GeneBank AAA21303). Preto:

representa 100% de identidade, amarelo: motivos Asp, rosa: motivo VTV, verde: sinal

de âncora GPI. No caso da gp82, o motivo VTV é subterminal e é detectado um

terceiro motivo Asp degenerado.

O clone J18 contém 2.139 pb e sua fase de leitura aberta (ORF) vai do

nucleotídeo 231 (ATG) ao 1779 (TGA), contendo 1548 nucleotídeos (Araya et

al., 1994). Codifica um polipeptídeo de 516 aminoácidos com massa molecular

de 55,6 KDa, menor do que a massa de 70 kDa do polipeptídeo sem os

oligossacarídeos N-ligados, precursor da gp82 (Ramirez et al., 1993). Para

tentar explicar essa discrepância, uma análise computacional apontou a

existência de diversos sítios de miristoilação, além dos sítios de N-glicosilação.

O tamanho dos mRNA transcritos a partir do gene gp82 foram compatíveis com

o da ORF clonada (Araya et al., 1994). Já o clone R31 contém 2.150 pb e sua

ORF vai do nucleotídeo 190 (ATG) ao 1786 (TGA), contendo 1596

nucleotídeos (Ruiz, 1998). Anterior à ORF há ainda 77 aminoácidos em fase de

leitura, sugerindo que a metionina encontrada pode não ser a primeira e que,

parte da região N-terminal da seqüência foi perdida. Sendo assim, R31 pode

ser considerado outro membro da família gp82 de maior tamanho em

comparação ao J18 (Ruiz, 1998).

Utilizando peptídeos sintéticos baseados na seqüência do clone J18,

Manque et al. (2000) mapearam o epítopo do MAb 3F6 (P3, 244-263) e o sítio

de adesão à célula hospedeira da gp82 de superfície, ambos presentes na

região do domínio central da molécula. O sítio de adesão celular é composto

pela justaposição de duas seqüências de aminoácidos separadas por outra

altamente hidrofóbica, e sobrepõe-se parcialmente ao epítopo do MAb 3F6 (P4,

254-273 e P8, 294-313) (Fig.19). Isso explica o fato desse anticorpo inibir a

invasão in vitro e in vivo de formas metacíclicas de cepas que utilizam

preferencialmente a gp82 (Ramirez et al., 1993). J18 e R31 possuem 100% de

identidade na seqüência de aminoácidos da região do sitio de adesão à célula

hospedeira (Yoshida, 2006).

Figura 19. Representação esquemática da gp82 de superfície de formas

metacíclicas do T. cruzi (clone J18).

Baseando-se na existência da família gênica gp82, nosso objetivo foi

caracterizar o repertório das moléculas gp82 expressas nas formas

metacíclicas, através da análise da expressão e localização celular nas cepas

G e CL. Para isso, clonamos um novo membro da família que, em contraste

com J18, não possui o epítopo do MAb 3F6, que caracteriza as moléculas gp82

de superfície. Os dados desse estudo mostraram que moléculas pertencentes

à família gp82 não são expressas somente na superfície das formas

metacíclicas, sugerindo outras funções além da adesão celular. Além disso,

observamos que a cepa G, juntamente com outras cepas do grupo T. cruzi I,

possui um conjunto de moléculas gp82 intraflagelares.

Experimentos complementares:

Análise da expressão de moléculas da família gp82 em formas

metacíclicas das cepas G e CL

Utilizando os anticorpos policlonais contra as proteínas recombinantes

J18 e C03, contendo ou não o epítopo do MAb 3F6, respectivamente,

analisamos os extratos de formas metacíclicas das cepas G e CL por

“immunoblot”. Na cepa CL, uma única banda foi detectada pelo MAb 3F6,

enquanto os anticorpos policlonais anti-J18 e anti-C03 reconheceram bandas

duplas (Fig.20A). Em “immunoblot” bidimensional, as moléculas reativas com o

MAb 3F6 focalizaram em pH 4.6 a 5.1 e as moléculas reativas com os

anticorpos anti-J18 e anti-C03, em pH 4.6 a 5.4 (Fig.20B). Na cepa G, ao

menos duas bandas foram reconhecidas por cada anticorpo (Fig.21A). Em

“immunoblot” bidimensional, as proteínas da família gp82 reativas com o MAb

3F6 ou com o anticorpo anti-J18, focalizaram em pH 4.9 a 5.7, e aquelas

reativas com o anticorpo anti-C03 focalizaram em pH 4.9 a 5.5 (Fig.21B).

Localização celular de moléculas gp82 reconhecidas pelo anticorpo

policlonal anti-C03 em cepas do grupo T. cruzi I ou II

Um resultado inesperado de nossos estudos foi a reação preferencial do

anticorpo anti-C03 com componentes intraflagelares da cepa G, o que não foi

observado na cepa CL (Fig. 4 e 5 do artigo). Como as cepas G e CL pertencem

a grupos genéticos divergentes, procuramos determinar se a localização

flagelar de proteínas gp82 reativas com o anticorpo anti-C03 seria uma

característica de cepas do grupo T. cruzi I, examinando duas outras cepas

deste grupo. Formas metacíclicas das cepas 262 (José-IMT) e 1161 (cedidas

pela Dra. Marta Teixeira, USP), permeabilizadas e incubadas com o anticorpo

anti-C03, apresentaram o mesmo perfil da cepa G, com marcação

predominantemente flagelar (Fig.22). Examinamos também formas

metacíclicas de outras duas cepas do grupo T. cruzi II, SC (Covarrubias et al.,

2007) e Y (Silva e Nussenzweig, 1953), quanto à reação com o anticorpo

policlonal anti-C03. Observamos padrão similar ao da cepa CL, com marcação

heterogênea no corpo do parasita (Fig.22). Concluímos que a localização

preferencial das proteínas gp82 no flagelo pode ser grupo-dependente.

Figura 20. Expressão de moléculas da família gp82 em formas metacíclicas da

cepa CL do T. cruzi. (A) Extratos dos parasitas foram analisados por “immunoblot”

com os anticorpos indicados. (B) “Immunoblot” bidimensionais de extratos de formas

metacíclicas mostrando proteínas separadas por focalização isoelétrica, seguida de

separação por SDS-PAGE e reação com os anticorpos indicados. Os números à

esquerda indicam as massas moleculares (kDa).

Figura 21. Expressão de moléculas da família gp82 em formas metacíclicas da

cepa G do T. cruzi. (A) Extratos dos parasitas foram analisados por “immunoblot” com

os anticorpos indicados. (B) “Immunoblot” bidimensionais de extratos de formas

metacíclicas mostrando proteínas separadas por focalização isoelétrica, seguida de

separação por SDS-PAGE e reação com os anticorpos indicados. Os números à

esquerda indicam as massas moleculares (kDa).

Figura 22. Localização celular de moléculas da família gp82 reconhecidas pelo

anticorpo policlonal anti-C03 em cepas do grupo T. cruzi I (262 e 1161) ou II (SC e

Y). Tripomastigotas metacíclicos vivos ou permeabilizados foram incubados com o

anticorpo anti-C03 e processados para visualização em microscópio de fluorescência.

Notar a localização predominantemente flagelar nas cepas do grupo T. cruzi I

permeabilizadas.

Expressão de moléculas da família gp82 em outras formas do T. cruzi

Os genes codificadores de proteínas dos tripanossomatídeos estão

organizados em unidades transcricionais policistrônicas, como ocorre nos

procariontes (Ullu et al., 1996). Sendo assim, mRNA de proteínas estágio-

específicas podem ser transcritos nas outras formas do parasita. Em análises

de transcrição do gene gp82, foram detectadas pequenas quantidades de

mRNA em epimastigotas, amastigotas e tripomastigotas (Carmo et al., 1999,

Manque et al., 2003, Songthamwat et al., 2007).

Restava saber se os transcritos gp82, presentes nas outras formas do

parasita além das formas metacíclicas, são traduzidos em proteínas. Para

responder a essa pergunta, analisamos a expressão de moléculas da família

gp82 nas formas epimastigotas, amastigotas e TCT, das cepas G e CL, por

“immunoblot” revelados com os anticorpos indicados (Fig.23).

Na cepa CL, o MAb 3F6 reagiu com moléculas gp82 somente das

formas metacíclicas, corroborando dados anteriores. O anticorpo policlonal

anti-J18, além das formas metacíclicas, reconheceu bandas de 60-100 kDa das

formas TCT, uma banda de ~110 kDa de amastigotas intracelulares e bandas

de ~70 e 82 kDa de amastigotas extracelulares. O anticorpo policlonal anti-

C03, além das formas metacíclicas, reconheceu bandas das formas TCT de

~82 e 95 kDa e não reconheceu nenhuma molécula de amastigotas (Fig.23A).

Na cepa G, o MAb 3F6 também reagiu com moléculas gp82 somente

das formas metacíclicas. O anticorpo policlonal anti-J18, além das formas

metacíclicas, reconheceu bandas de 67-100 kDa das formas TCT e bandas de

~70 e 82 kDa de amastigotas extracelulares. O anticorpo policlonal anti-C03,

além das formas metacíclicas, reconheceu uma banda de ~95 kDa das formas

TCT e uma banda de ~67 kDa de formas amastigotas extracelulares (Fig.23B).

Os dados mostram que moléculas da família gp82 sem o epítopo do

MAb 3F6 são expressas em todas as formas do T. cruzi, exceto em

epimastigotas. Porém, a expressão é diferencial entre as cepas G e CL. Além

disso, os anticorpos policlonais anti-J18 e anti-C03, apesar de apresentarem

reatividade cruzada (Fig.3 do artigo), reagem com diferentes membros da

família gp82 nas formas do parasita analisadas.

A Cepa CL

B Cepa G

Figura 23. Expressão de moléculas da família gp82 em diferentes formas do T.

cruzi. Extratos dos parasitas foram analisados por “immunoblot” com os anticorpos

indicados. (A) Cepa CL. (B) Cepa G. E, epimastigotas, M, formas metacíclicas, T,

tripomastigotas de cultura de tecido, AI, amastigotas intracelulares, AE, amastigotas

extracelulares. Os números à esquerda indicam as massas moleculares (kDa).

Capítulo 3

Indução de apoptose em células de melanoma pela

gp82 em sua forma recombinante

Artigo:

“A recombinant protein based on Trypanosoma cruzi surface molecule

gp82 induces apoptotic cell death in melanoma cells”

Atayde, V., Jasiulionis, M.G., Cortez, M., Yoshida, N.

Melanoma Research, 2008, 18: 172-183

O melanoma inicia-se a partir da transformação maligna do melanócito,

célula produtora de pigmento encontrada na lâmina basal da epiderme. Essa

transformação ocorre devido a diversos fatores genéticos, epigenéticos e

ambientais (Satyamoorthy & Herlyn, 2002, Correa et al., 2005). Normalmente,

os melanócitos sintetizam melanina e a transferem em grânulos aos

queratinócitos subjacentes, que a armazenam. Essa pigmentação protege

contra os danos causados pela radiação ultravioleta solar (Ueda & Richmond,

2006) (Fig.24).

Figura 24. Melanócitos na lâmina basal da epiderme. Em destaque está a

transferência de grânulos de melanina aos queratinócitos (training.seer.cancer.gov).

A linhagem de melanócitos melan-a, derivada de camundongo C57BL,

possui características de melanócitos normais, exceto senescência e resposta

proliferativa ao PMA (phorbol-12-myristate-13-acetate), ativador da PKC

(Bennett et al., 1987, Furstenberger et al., 1981). A partir de melan-a, foi

estabelecida a linhagem de melanoma Tm5, que apresenta crescimento

independente de PMA, menores tempos de duplicação e diferente adesividade

a componentes da matriz extracelular. Além disso, Tm5 é altamente

tumorigênica in vivo (Oba-Shinjo et al., 2006).

A incapacidade das células em entrar em apoptose (Fig.25) contribui

para a patogênese de diversos cânceres, incluindo o melanoma (Hanahan &

Weinberg, 2000, Herlyn et al., 2002).

Figura 25. Vias clássicas de indução da apoptose. Na via extrínseca, é ativada a

caspase-8. Na via intrínseca, é ativada a caspase-9. IAP, c-FLIP, Bcl-2, HSP70 e

HSP90 são inibidores endógenos da apoptose. Bax e p53 são indutores (adaptado de

Heussler et al., 2001).

A morte celular por apoptose é induzida por duas vias principais: uma

extrínseca, que envolve os membros da família de receptores do TNF (fator de

necrose tumoral) com recrutamento e ativação da caspase-8, e outra

intrínseca, através da liberação do citocromo C mitocondrial e sua interação

com Apaf-1 no citoplasma, com ativação da caspase-9 (Fig.25). As duas vias,

que possuem conexões entre si (Taylor et al., 2008), convergem para ativar as

caspases efetoras 3, 6 e 7, responsáveis pelas alterações específicas da

apoptose: condensação e clivagem do DNA, exposição da fosfatidilserina na

porção externa da membrana plasmática e fragmentação celular em corpos

apoptóticos (Fig.26), dentre outras (Heussler et al., 2001, Fulda & Debatin,

2006). A liberação do citocromo C é controlada pela família bcl-2 de proteínas,

que tem sua transcrição regulada pelo fator NF-κB, constitutivamente ativo em

células de melanoma (Richmond, 2002, Fecker et al., 2005, Amiri and

Richmond, 2005, Ueda and Richmond, 2006).

Figura 26. Corpos apoptóticos observados por microscopia eletrônica de

varredura. Os corpos apoptóticos não permitem o extravasamento do conteúdo

intracelular no tecido, protegendo-o de inflamação (mun.ca).

Durante a apoptose, o citoesqueleto de actina, responsável pelo formato,

motilidade celular e transdução de sinais exteriores (Ho et al., 2008), é alvo das

caspases efetoras. Mudanças na dinâmica da actina filamentosa (F-actina), por

sua vez, induzem a ativação das caspases (Leadsham & Gourlay, 2008).

O aumento do “turnover” da F-actina promove a longevidade celular,

enquanto que a diminuição pode induzir a apoptose (Gourlay & Ayscough,

2005, Thomas et al., 2006). Drogas que alteram a dinâmica da F-actina, como

a citocalasina D, ativam a apoptose em células humanas e murinas através de

uma via caspase-3-dependente (Posey & Bierer, 1999, Yamazaki et al., 2000,

Odaka et al., 2000, Boldogh & Pon, 2006). Ainda não se sabe ao certo por qual

mecanismo a caspase-3 é ativada nesses casos. A proteína recombinante J18,

baseada na molécula de superfície gp82 de formas metacíclicas do T. cruzi

(Araya et al., 1994), induz despolimerização do citoesqueleto de actina, efeito

análogo ao da citocalasina D (Cortez et al., 2006b) (Fig.27). Esse efeito é

dependente da liberação de Ca

induzida por J18 na célula hospedeira (Ruiz

et al., 1998, Cortez et al., 2006b).

Figura 27. Diminuição das fibras de actina em células HeLa tratadas com J18 ou

citocalasina D por 30 minutos. Verde: actina, azul: núcleos (Cortez et al., 2006b).

As interações entre a actina e as mitocôndrias, que agem como

sensores da diminuição do “turnover” da actina, são centrais na regulação da

apoptose pelo citoesqueleto. (Gourlay and Ayscough, 2005, Leadsham &

Gourlay, 2008). Quando a actina é estabilizada, um canal iônico voltagem-

depentente (VDAC) da mitocôndria é aberto, permitindo a liberação de

moléculas apoptogênicas como citocromo C, AIF e smac (Gourlay & Ayscough,

2005). Além disso, alguns membros da família bcl-2 podem interagir

diretamente com VDAC, determinando a permeabilidade da membrana

mitocondrial (Tsujimoto, 2002). Transições na permeabilidade alteram o

potencial transmembrânico, um dos primeiros sinais apoptóticos de células de

mamíferos (Simeonova et al., 2004) (Fig.28).

Figura 28. Regulação de VDAC pela actina. A redução na dinâmica da actina pode

levar à redução no potencial mitocondrial e indução da apoptose após a liberação de

fatores apoptogênicos (adaptado de Gourlay & Ayscough, 2005).

Muitas evidências experimentais mostram que infecções com diversos

patógenos, como Salmonella typhimurium (Pawelek et al., 1997), Toxoplasma

gondii (Hunter et al., 2001) ou Trypanosoma cruzi (Oliveira et al., 2001), podem

retardar ou inibir o crescimento de tumores malignos, inclusive dos melanomas,

em animais e humanos. Para explicar o efeito antitumoral das infecções pelo T.

cruzi, alguns pesquisadores propuseram a existência de uma toxina secretada

pelos parasitas, outros a ocorrência de resposta imune contra antígenos das

células tumorais (Cabral, 2000).

Pouco se sabe sobre a produção de fatores solúveis ou secretados por

patógenos que podem agir nas células tumorais, causando sua morte. A

proteína azurina, de Pseudomonas aeruginosa, entra em células de melanoma

humano e induz apoptose devido ao aumento dos níveis intracelulares de p53

(Yamada et al., 2002). Uma fração rica em lipídeos, isolada de promastigotas

de Leishmania donovani, induz apoptose em melanomas de camundongo e

humano (Ratha et al., 2006).

Evidências sobre as propriedades antitumorais do T. cruzi mostram o

efeito citotóxico direto de lisados dos parasitas sobre células tumorais in vitro

(Kallinikova et al., 2001, Sheklakova et al., 2003), efeito que deve existir devido

ao potencial pró-apoptótico que é visto em algumas moléculas do T. cruzi

contra células do sistema imune. Uma dessas moléculas é a transialidase

(Freire-de-Lima et al., 1998, Borges et al., 2005, Mucci et al., 2006).

Nosso principal objetivo foi investigar o efeito pró-apoptótico da proteína

gp82 recombinante (J18) sobre células do melanoma Tm5, comparado ao

efeito sobre a linhagem de melanócitos melan-a. Os resultados mostraram que

o tratamento com J18 induz a despolimerização da F-actina somente nas

células do melanoma Tm5, levando à indução da apoptose através da ativação

da caspase-3. Sinais apoptóticos específicos como encolhimento nuclear e

citoplasmático, exposição da fosfatidilserina e fragmentação do DNA nuclear,

foram observados. Também notamos a perda do potencial transmembrânico e

a localização citoplasmática de NF-kB decorrentes do efeito de J18 no

citoesqueleto de actina.

Experimentos complementares:

Análise da proteína recombinante J18:

Uma questão em relação à proteína recombinante J18 que não havia sido

ainda determinada é como ela se apresenta na forma purificada, se como

monômero, dímero ou aglomerado protéico. Para responder a essa pergunta,

purificamos uma preparação de J18 por gel filtração em sistema FPLC e

obtivemos três principais picos correspondentes a: proteínas maiores de 160

KDa representando aglomerados de J18, proteínas de aproximadamente 160

kDa representando dímeros de J18, proteínas de aproximadamente 80 kDa

representando monômeros de J18 (Fig.29A). Submetemos as frações 11 a 43 à

SDS-PAGE em condições desnaturantes, seguido de “immunoblot” revelado

com o MAb 3F6. Como mostrado na Figura 29B, bandas de aproximadamente

80 kDa foram detectadas nas frações 14 a 30 e uma maior quantidade de

proteína foi detectada nas frações 20 a 24, representando aglomerados e

dímeros de J18 (>160 kDa e ~160 kDa). Selecionamos algumas frações

representantes dos diferentes picos da gel filtração e submetemos à eletroforese

não-desnaturante seguida de “immunoblot” e revelação com o MAb 3F6. Foram

detectadas bandas maiores de 80 kDa, atingindo até ~230 kDa, principalmente

nas frações 21 e 24, representantes dos picos de proteínas maiores de 160 kDa

e de ~160 kDa (Fig.30).

O efeito inibitório de J18 sobre a invasão celular de formas metacíclicas é

conhecido. Para comparar com o efeito de J18 na sua forma monomérica,

dimérica ou oligomérica, realizamos experimentos com as seguintes

preparações: frações 11, 21, 24 e 27, a preparação completa de J18, o

sobrenadante e o “pellet” de uma preparação de J18 centrifugada a 13.000 rpm

por 15 minutos. A inibição da invasão foi semelhante (~40%) para todas as

amostras, exceto para a fração 11, que foi utilizada como controle (Fig.31).

Figura 29. Purificação da proteína recombinante J18 por gel filtração em sistema

FPLC. (A) Uma preparação de J18 foi purificada em coluna cromatográfica de gel

filtração Superdex HR200 em sistema FPLC, como descrito (Amino et al., 2002). (B)

As frações de 0.5 ml coletadas foram submetidas à SDS-PAGE e “immunoblot”

revelado com o MAb 3F6. Notar o reconhecimento das frações 14 a 30 pelo MAb 3F6,

de acordo com os picos do gráfico.

Figura 30. “Immunoblot” de preparações da proteína recombinante J18

submetidas à SDS-PAGE não-desnaturante. As frações coletadas da gel filtração

(11, 21, 24 e 27), a proteína total (J18), a proteína centrifugada (C), ou o “pellet” (P)

foram submetidos à SDS-PAGE não-desnaturante, transferidos à membrana de

nitrocelulose e revelados com o MAb 3F6. Notar o reconhecimento de bandas maiores

de 80 kDa principalmente nas frações 21 (> 160 kDa) e 24 (~160 kDa). Os números à

esquerda representam as massas moleculares (kDa).

Figura 31. Invasão celular de formas metacíclicas da cepa CL na presença de

preparações da proteína recombinante J18. Formas metacíclicas da cepa CL foram

incubadas por 1 hora com células HeLa (10: 1) na presença das frações coletadas da

gel filtração (11, 21, 24 e 27), da proteína total (J18), da proteína centrifugada (C), ou

do “pellet” da centrifugação (P), na concentração final de 40 µg/ml. O total de parasitas

em 100 células foi estimado contando-se lamínulas coradas com Giemsa em

duplicatas (500 células por lamínula). Experimento representativo de três

experimentos independentes.

Com esses resultados, mostramos que uma preparação de J18 contém

proteínas com diferentes níveis de agregação e que essas diferentes formas têm

a mesma capacidade de interagir com a célula hospedeira, competindo com os

parasitas no momento da invasão. Dessa maneira, obtivemos mais informações

em relação ao preparado (J18) utilizado no tratamento das células melan-a e

Tm5.

Imagens de tumores em camundongos infectados com T. cruzi

Camundongos C57BL/6 foram infectados com formas metacíclicas de

duas cepas de T. cruzi expressando ou não a gp82 na superfície, cepas G e Y*,

respectivamente. Três semanas após a infecção, os camundongos receberam

células do melanoma Tm5 (2 x 10

) por via subcutânea na região dorsal e o

desenvolvimento dos tumores foi acompanhado. Os volumes tumorais foram

significativamente menores nos animais infectados com a cepa G, comparados

aos volumes tumorais dos camundongos infectados com a cepa Y* ou dos

camundongos não-infectados, como mostrado na figura 9 do artigo (Fig.32).

Figura 32. Desenvolvimento tumoral (20 dias pós-inoculação de células Tm5) em

camundongos infectados com T. cruzi.

Discussão

Nosso principal objetivo foi caracterizar a família gp82 de proteínas do T.

cruzi. Até o início desse projeto, somente a gp82 de superfície, identificada pelo

MAb 3F6 e envolvida na invasão celular das formas metacíclicas, havia sido

descrita. Mostramos que membros da família gp82 são expressos

intracelularmente e que alguns têm localização diferenciada em parasitas dos

grupos T. cruzi I e II. Ao contrário das glicoproteínas gp82 reativas com o MAb

3F6, específicas das formas metacíclicas (Teixeira & Yoshida, 1986), outras

moléculas da família foram detectadas nas formas TCT e amastigotas.

Descrevemos ainda o efeito antitumoral da gp82 de superfície em sua forma

recombinante, propriedade nunca antes descrita para outro componente do T.

cruzi molecularmente definido.

Na primeira parte do estudo, investigamos as bases da avirulência das

formas metacíclicas do clone CL-14, derivado da cepa CL. Descobrimos que a

baixa infectividade desses parasitas está associada com a reduzida expressão

da gp82 na superfície, reforçando o papel central da molécula na infecção pelo

T. cruzi. Além de invadir cerca de quatro vezes menos do que a cepa CL in

vitro, as formas amastigotas do clone CL-14 não foram capazes de se replicar

em células HeLa mantidas a 37

C. Esse resultado está de acordo com achados

anteriores onde foi mostrado que a temperatura permissiva para a

diferenciação do clone CL-14 em células não-fagocíticas é de 33

C (Almeida-

de-Faria et al., 1999). A temperatura como um fator do hospedeiro na inibição

do desenvolvimento intracelular é compatível com nossa observação de que as

formas metacíclicas do clone CL-14 não produzem parasitemia patente em

camundongos infectados por via oral.

Uma vez que a infecção oral por formas metacíclicas da cepa CL é

extremamente eficiente e é dependente da gp82 de superfície (Neira et al.,

2003), a deficiência na expressão dessa molécula no clone CL-14 seria o único

fator responsável pela baixa infectividade, não fosse seu desenvolvimento

limitado pela temperatura. Na infecção oral, além de promover a invasão das

células da mucosa gástrica, foi descrito que a interação entre a gp82 e a

mucina é fundamental (Neira et al., 2003). No estudo de formas metacíclicas de

cepas do T. cruzi gp82-deficientes, observou-se que essas são pouco

infectivas em camundongos por via oral, embora a invasão celular in vitro seja

eficiente devido à expressão da glicoproteína gp30, que tem características

similares à gp82 (Cortez et al., 2003). Gp30 liga-se à célula hospedeira de

maneira receptor-dependente e induz sinal de Ca

, propriedade associada à

invasão celular. Entretanto, ao contrário da gp82, a capacidade de ligação da

gp30 à mucina gástrica é reduzida (Cortez et al., 2003). No caso do clone CL-

14, as formas metacíclicas não expressam na superfície nem a gp82 nem a

gp30, e são pouco invasivas tanto in vivo como in vitro.

As formas metacíclicas do clone CL-14 e da cepa G assemelham-se

quanto à baixa infectividade e aparentemente prescindem da gp82 para invadir

a célula hospedeira. É possível que o clone CL-14, como a cepa G, faça uso da

gp35/50 para interagir com a célula hospedeira. Entretanto, a gp35/50 expressa

no clone CL-14 é uma variante da gp35/50 da cepa G, que reage com o MAb

2B10 mas não é reconhecida pelo MAb 10D8 (Mortara et al., 1992). Embora as

formas metacíclicas da cepa G expressem gp82 na superfície em níveis

comparáveis aos da cepa CL, a interação da gp82 com a célula hospedeira é

prejudicada pela gp90, molécula que modula negativamente a penetração do

parasita (Málaga e Yoshida, 2001). O clone CL-14 expressa baixos níveis de

uma molécula variante da gp90 da cepa G, reconhecida pelo MAb 5E7, mas

não pelo MAb 1G7 (Yoshida, 2006). Nesse caso, a gp90 provavelmente não

atua como modulador negativo da invasão.

Na segunda parte do estudo, analisamos a expressão e a localização de

moléculas da família gp82 em formas metacíclicas das cepas G e CL. Para

isso, isolamos um novo membro, que não contém o epítopo do MAb 3F6.

Quando alinhamos as seqüências de aminoácidos dos clones C03 (gp82) e Tc-

85 (gp85 das formas TCT), observamos 57,2% de identidade, indicando que o

novo clone é parte da família gp85/trans-sialidase. As duas seqüências

possuem semelhanças na região N-terminal, que é inexistente no clone J18

(gp82). J18 e Tc-85 apresentam 53,3% de identidade (Fig.33).

Figura 33. Alinhamento das seqüências de aminoácidos dos clones J18, C03 e

Tc85. J18 (AAA21303), C03 (EF445668) Tc85-11 (AAD13347.1). Preto: representa

100% de identidade, amarelo: motivos Asp, rosa: motivo VTV, verde: sinal de âncora

GPI, vermelho: regiões do epítopo do MAb 3F6 e do sítio de adesão celular do clone

J18, azul: sítio de ligação à laminina do clone Tc-85.

Um achado interessante desse trabalho foi a marcação

predominantemente flagelar do anticorpo policlonal anti-C03 em formas

metacíclicas de cepas do grupo T. cruzi I. O flagelo dos tripanossomatídeos é

único e multifuncional, atuando na motilidade, quimiotaxia, sinalização e

invasão celular. Muitas moléculas do T. cruzi associadas ao flagelo têm sido

descritas. Uma delas, cuja função ainda não se conhece, tem 160 kDa (FL-160)

e é codificada por uma família gênica que é parte da grande família gp85/trans-

sialidase. Essa proteína é expressa em formas TCT, na membrana do bolso

flagelar, região de onde emerge o flagelo (Van Voorhis et al., 1993). Um

membro dessa família gênica, FL-160-CRP, é expresso na superfície dos

parasitas, mas não é expresso no flagelo. Na análise dos transcritos da FL-

160-CRP, variações nas seqüências foram associadas com a divisão das

cepas em grupos T. cruzi I e II (Mathieu-Daudé et al., 2008). Se o anticorpo

anti-C03 pode ser usado como um marcador de cepas do grupo T. cruzi I,

ainda precisa ser estabelecido. A função dos membros da família gp82

expressos no flagelo é desconhecida.

Em comparação com o clone J18, os clones C03 e Tc-85 apresentam

diversas substituições de aminoácidos nas regiões do epítopo do MAb 3F6 e

do sítio de adesão celular da gp82 (Fig.33). Talvez por isso a gp85 não tenha

sido reconhecida pelo anticorpo monoclonal em extratos de formas TCT das

cepas G ou CL, nos ensaios de “immunoblot”. Porém, como possuem diversos

epítopos comuns, os anticorpos policlonais anti-J18 e anti-C03 reagiram com

um conjunto de moléculas dessas formas, que possivelmente inclui a gp85

(Fig.23).

Em relação à adesão celular, foi descrito que a molécula de superfície

gp85 contém sítios de ligação à laminina e à citoqueratina 18 (CK18) (Giordano

et al., 1999, Magdesian et al., 2001), interações que podem promover ou

facilitar a invasão das formas TCT. Utilizando peptídeos sintéticos, Giordano et

al. (1999) mapearam o sítio de adesão do clone Tc-85 à laminina (peptídeo

TGETPLEPFGFCFGA). Dos 15 aminoácidos que compõem a região referente

ao peptídeo de ligação à laminina, o clone J18 possui apenas 5 em comum,

sugerindo que se essa molécula também interage com a laminina é através de

outro sítio (Fig.33). A Tc-85 interage com a CK18 através do motivo VTV, que

aumenta a invasão das formas TCT (Magdesian et al., 2007). O clone J18

possui esse motivo na região subterminal (Fig.33), e a sua relevância na

adesão do parasita à célula é discutível.

Outras moléculas das formas TCT, além da gp85, interagem direta ou

indiretamente com moléculas da matriz extracelular (ECM). A gp83, molécula

de superfície de 83 kDa, aumenta a expressão da laminina-γ1 em células

musculares humanas após a exposição à sua forma recombinante. A

superexpressão da laminina torna as células mais susceptíveis à infecção (Nde

et al., 2006). A gp83 tem 32,8% de identidade na seqüencia de aminoácidos

com o clone J18 e 32,3% com o clone C03. É possível que os anticorpos

policlonais anti-C03 e anti-J8 reconheçam epítopos da gp83 das formas TCT

das cepas G ou CL, como visto na figura 23. Foram também detectadas, nas

formas TCT, proteínas ligantes de heparina e heparan sulfato de

aproximadamente 59 e 65,8 kDa, e essa ligação está envolvida na invasão de

cardiomiócitos. Uma delas pode ser a penetrina, previamente descrita, que

possui as mesmas características (Ortega-Barria & Pereira, 1991, Oliveira Jr. et

al., 2008). A serino-protease POP Tc80, hidrolisa colágeno dos tipos I e IV e

fibronectina, provavelmente como modo de facilitar a migração através da ECM

(Grellier et al., 2001, Bastos et al., 2005). Também foi mostrado que formas

TCT possuem um receptor de superfície para a fibronectina (Ouaissi et al.,

1984).

A ligação de moléculas do T. cruzi à laminina, heparan sulfato,

fibronectina e outros componentes, pode ser essencial na migração dos

parasitas através da ECM em direção às células-alvo. É importante lembrar

que, de maneira similar, a gp82 interage com a mucina gástrica, facilitando o

acesso das formas metacíclicas às células do epitélio gástrico durante a

infecção oral (Neira et al., 2003). Até o momento, o receptor celular da gp82 de

superfície não foi identificado. Além disso, a qual componente da ECM se liga a

gp82 também não é conhecido. Foi mostrado que formas TCT entram em

células epiteliais preferencialmente pela porção baso-lateral (Schenkman et al.,

1991c). Se isso ocorrer também com as formas metacíclicas, uma hipótese

seria que a gp82 contém sítios de interação com componentes da lâmina basal,

já que essas formas, quando iniciam a infecção no hospedeiro vertebrado,

invadem células epiteliais. A gp82 parece não interagir com colágeno tipo IV,

presente na lâmina basal (Ramirez et al., 1993). Resta saber se interage com a

laminina.

Na terceira parte do estudo, ocorreu-nos a idéia de examinar a ação de

J18 sobre as células de melanoma, em função das propriedades dessa

molécula reveladas em trabalhos anteriores. Cortez et al. (2006b) observaram

que J18 induz a despolimerização da F-actina de células HeLa. Levantamos a

possibilidade de que o tratamento das células com J18 por tempo prolongado

levaria, em última instância, à morte celular. Nos diversos experimentos, J18

mostrou efeito seletivo sobre as células do melanoma Tm5, mas não sobre as

células parentais não tumorigênicas, melan-a. Esse efeito não se deve à falta

do receptor para essa molécula nas células melan-a, pois J18 liga-se a estas

células do mesmo modo que às células Tm5. Uma possibilidade é que, após a

ligação de J18 ao seu receptor, vias de sinalização distintas sejam induzidas

nas duas linhagens, levando à desestruturação dos filamentos de actina

somente nas células de melanoma (Fig.34). Em células HeLa, foi mostrado que

a despolimerização da F-actina é dependente de sinal de Ca

, deflagrado

após a ativação de uma via de sinalização ainda desconhecida (Cortez et al.,

2006b).

Segundo dados da literatura, a citocalasina D induz morte celular por

apoptose em células transformadas através da ativação da caspase-3

(Yamazaki et al., 2000) sendo estas mais susceptíveis do que células normais

(Odaka et al., 2000). O fato das células tumorais conterem menor quantidade

de F-actina do que células e tecidos normais (Verderame et al., 1980, Varani et

al, 1983, Rao et al., 1990), poderia explicar a diferença entre as

susceptibilidades de melan-a e Tm5 à J18. Como J18 tem ação similar à da

citocalasina D (Cortez et al., 2006b), analisamos o tipo de morte induzida por

esta molécula nas células Tm5 e encontramos diversas evidências de

apoptose. O primeiro evento apoptótico detectado foi a ativação da caspase-3,

provavelmente responsável pela ocorrência dos eventos subseqüentes

(Fig.34). Não observamos ativação das caspases 8 e 9. Algumas moléculas do

T. cruzi apresentam efeito contrário ao de J18. A transialidase inibe a apoptose

em células de Schwan humanas, através de uma via ativada pela PI-3 quinase

(Chuenkova et al., 2001). A cruzipaína, cisteína protease envolvida na invasão,

crescimento e diferenciação do T. cruzi (Murta et al., 1990, Paiva et al., 1998,

Meirelles et al., 1992, Scharfstein et al., 2000), quando inativada, promove a

sobrevivência de cardiomiócitos de culturas primárias de camundongos através

da indução da expressão de Bcl-2, molécula anti-apoptótica (Aoki et al., 2006).

Figura 34. Representação esquemática da via apoptótica ativada em células do

melanoma Tm5 pelo tratamento com J18. A ligação de J18 com seu receptor (J18R)

na célula Tm5 leva à desorganização do citoesqueleto de actina com ativação da

caspase-3, responsável pela exposição da fosfatidilserina (PS), perda do potencial da

membrana mitocondrial (ψm), inibição da translocação de NF-κB ao núcleo e

fragmentação do DNA nuclear, fenômenos apoptóticos. Entre parênteses estão os

tempos de tratamento.

Uma possibilidade que não foi confirmada em nossos estudos é a

ativação da caspase-3 nas células Tm5, via ativação da caspase-12. A caspase-

12, que está envolvida na indução da apoptose após estresse de retículo

endoplasmático (Liu et al., 1998, Rao et al., 2002, Xie et al., 2002, Hitomi et al.,

2003) poderia, em seguida, ativar a caspase-3 nas células Tm5. Outra molécula

que poderia estar envolvida na ativação da apoptose em células Tm5 é a

calpaína, cisteína protease citossólica também intimamente relacionada com a

via de estresse de retículo endoplasmático. Essa enzima é ativada por Ca

tem como alvo a caspase-12, ativando-a (Vandenabeele et al., 2005).

Investigamos ainda o efeito tumoricida de J18 in vivo. O

desenvolvimento do melanoma Tm5 foi significativamente mais lento em

camundongos tratados com J18 na região tumoral. Eventualmente, esses

resultados poderiam ser melhorados com outras dosagens de J18 e/ou tempo

de tratamento. O bloqueio da translocação de NF-kB ao núcleo in vitro pode ser

também a causa da inibição do crescimento de células Tm5 in vivo. Esse

resultado contrasta com resultados recentes com a transialidase em sua forma

recombinante que, ao invés de inibir, ativa a translocação de NF-kB ao núcleo

de células endoteliais humanas. Esse resultado é também observado quando

as células são tratadas com lipopolissacarídeo (LPS) bacteriano (Dias et al.,

2008). Contudo, não podemos descartar a hipótese de que o J18 tenha outra

ação in vivo, diferente da observada in vitro. Talvez o crescimento dos tumores

seja reduzido pela indução de alterações importantes no microambiente

tumoral que, por sua vez, eliminariam as células tumorais dos tecidos.

Quanto à proteção ao desenvolvimento tumoral pela infecção com o T.

cruzi, descrita em diversos trabalhos, confirmamos esses achados infectando

camundongos com a cepa G, que é pouco infectiva, porém expressa altos

níveis de gp82 na superfície, e com a cepa Y*, uma amostra da cepa Y, isolada

inicialmente por Silva & Nussenzweig (1953) de um paciente na fase aguda da

doença de Chagas. Formas metacíclicas de Y* expressam somente a gp30 na

superfície. Apesar das formas metacíclicas existirem por pouco tempo no

hospedeiro vertebrado, observamos significativa redução tumoral em

camundongos pré-infectados com a cepa G. Em contraste, camundongos pré-

infectados com a cepa Y*, que não expressa a gp82 reativa com o MAb 3F6,

mas expressa outros membros da família gp82 reconhecidos pelo anticorpo

policlonal anti-J18, foram incapazes de conter o crescimento tumoral.

A associação entre esses dados e os dados referentes à gp82

recombinante (J18) não é clara. O que pode ser hipotetizado é que moléculas

da família gp85/trans-sialidase, que têm seqüências em comum com a gp82, e

que são expressas em tripomastigotas sanguíneos, exerçam o papel de reduzir

o desenvolvimento dos tumores. Para citar um exemplo, a trans-sialidase

presente na corrente sanguínea, secretada pelos parasitas, possui a

característica de induzir a apoptose de células do sistema imune do hospedeiro

(Mucci et al., 2006). Outra molécula que poderia ser responsável pela redução

tumoral é a gp85 expressa pelas formas sanguíneas da cepa G, uma vez que

vimos que as duas moléculas, gp82 e gp85 possuem epítopos comuns. Seria

interessante investigar se a gp85 de fato possui efeito antitumoral como

observamos com a gp82 recombinante. Outra explicação para a redução

tumoral em camundongos infectados com a cepa G seria a de que membros da

família gp82 expressos por formas sanguíneas ou amastigotas, quando

liberados no meio extracelular ou na corrente sanguínea, pudessem agir nas

células tumorais. Os membros da família gp82 expressos por formas

metacíclicas da cepa Y* não teriam função antitumoral.

Segundo Cabral (2000), a redução dos tumores de camundongos

infectados com T. cruzi ocorreria pelo desenvolvimento de uma resposta auto-

imune contra as células tumorais, devido à lise daquelas infectadas com o

parasita. Essa resposta ocorreria devido ao extravasamento e exposição do

conteúdo celular no meio extracelular. Alguns estudos mostram a auto-

imunidade à miosina cardíaca desenvolvida em camundongos ou humanos

infectados com T. cruzi. Porém, não se sabe ao certo se é resultado da lise e

destruição celular, ou da reatividade cruzada entre antígenos do parasita e do

hospedeiro (Engman & Leon, 2002). Dessa maneira, existe a possibilidade de

ocorrer reatividade cruzada entre moléculas dos parasitas da cepa G e das

células tumorais.

Nossos estudos abrem perspectivas para o estudo das proteínas da

família gp82, principalmente dos membros que possam ter funções diferentes

da função da gp82 de superfície, como por exemplo, os flagelares. Em

trabalhos subseqüentes, esses membros devem ser caracterizados

funcionalmente.

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Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

Vanessa D. Atayde

, Ivan Neira

, Mauro Cortez

, Daniele Ferreira

Edna Freymu

ller

, Nobuko Yoshida

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de Sa

o Paulo,

R. Botucatu, 862-6º

andar, Sa

o Paulo, SP 04023-062, Brazil

Centro de Microscopia Eletro

nica, Escola Paulista de Medicina, Universidade Federal de Sa

o Paulo,

R. Botucatu, 862-6º

andar, Sa

o Paulo, SP 04023-062, Brazil

Received 12 January 2004; received in revised form 5 March 2004; accepted 5 March 2004

Abstract

We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental

isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent

infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold

lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL

isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the

kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very

close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The

surface proﬁle was similar, except that the expression of gp82, the stage-speciﬁc glycoprotein that promotes CL isolate mucosal infection in

vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a speciﬁc inhibitor of

protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-

14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a

combination of ionomycin plus NH

Cl, which releases Ca

2þ

from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was

signiﬁcantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50.

Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of

gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.

Keywords: Trypanosoma cruzi; Clone CL-14; Gp82; Metacyclic trypomastigotes; Cell invasion; Signal transduction

1. Introduction

The virulence of Trypanosoma cruzi, the protozoan

parasite that causes Chagas’ disease, is associated with its

ability to invade host cells and to replicate. Invasion of

mammalian cells by T. cruzi is a multi-step process that

requires the interaction of parasite and target cell molecules

and activation of signal transduction pathways in both cells.

Metacyclic trypomastigotes, the developmental forms

responsible for initiating T. cruzi infection in the mamma-

lian host, engage the stage-speciﬁc surface glycoprotein

gp82 to establish the initial parasite– host cell interaction

and promote invasion (Ramirez et al., 1993; Ruiz et al.,

1998). Binding of gp82 triggers the signaling cascades in the

parasite as well as in the target cell, leading to Ca

2þ

mobilisation (Ruiz et al., 1998; Yoshida et al., 2000), which

is an essential requirement for parasite internalisation

(Moreno et al., 1994; Tardieux et al., 1994; Dorta et al.,

1995).

Experiments in mice have indicated that metacyclic

trypomastigotes have the uniquely specialised properties of

gastric mucosal invasion upon oral challenge (Hoft, 1996;

Hoft et al., 1996), a route to which is attributed the

microepidemics responsible for more than half of the acute

cases of Chagas’ disease recorded between 1968 and 2000

in Brazilian Amazon (Coura et al., 2002). Recent studies

have shown that stage-speciﬁc gp82 plays a central role in

doi:10.1016/j.ijpara.2004.03.003

International Journal for Parasitology 34 (2004) 851–860

www.parasitology-online.com

Corresponding author. Tel.: þ55-11-5576-4532; fax: þ 55-11-5571-

1095.

E-mail address: [email protected] (N. Yoshida).

mucosal invasion of metacyclic forms, leading to systemic

infection upon oral inoculation (Neira et al., 2003).

T. cruzi isolate CL, which is highly infective in vitro and

in vivo (Yoshida, 1983; Ruiz et al., 1998; Neira et al., 2003)

has been used in experiments to establish the role of gp82 in

host cell invasion (Ruiz et al., 1998; Favoreto et al., 1998;

Yoshida et al., 2000). What is intriguing is that CL-14, a

clone derived from the CL isolate, is unable to produce a

patent infection even when injected into new-born mice

(Lima et al., 1990), negative parasitism resulting from

extensive histopathological analysis of mouse tissues and

organs, at different times after intraperitoneal or intravenous

injection of metacyclic forms (Lima et al., 1995). As

different T. cruzi isolates may differentially engage their

repertoire of surface molecules to interact with host cells,

activating distinct signal transduction pathways that deter-

mine the efﬁciency of parasite internalisation (Ruiz et al.,

1998; Neira et al., 2002), the deﬁcient infectivity of clone

CL-14 could be associated with its surface proﬁle.

Investigating the molecular basis of lack of virulence of

clone CL-14 may be important to further clarify the

complex process of T. cruzi infection. To this end, we

analysed in this study the properties of metacyclic forms of

clone CL-14, as compared to those of CL isolate, focusing

on the proﬁle of surface molecules, gp82 in particular; the

activities of cruzipain and trans-sialidase, enzymes that

have been implicated in target cell penetration (Meirelles

et al., 1992; Schenkman et al., 1991); and the ability to

invade epithelial HeLa cells upon treatment of parasites

with drugs that interfere with signal transduction and

intracellular Ca

2þ

mobilisation.

2. Materials and methods

2.1. Trypanosoma cruzi, mammalian cells

and cell invasion assay

The T. cruzi isolate CL (Brener and Chiari, 1963) was

maintained cyclically in mice and in axenic culture in liver

infusion tryptose (LIT) medium and the clone CL-14

(Chiari, 1981) was grown in LIT medium throughout this

study. To obtain cultures enriched in metacyclic trypomas-

tigotes, Grace’s medium (Life Technologies) was also used.

Metacyclic forms from cultures at the stationary growth

phase were puriﬁed by passage through DEAE-cellulose

column, as described (Teixeira and Yoshida, 1986). HeLa

cells, the human carcinoma-derived epithelial cells, were

grown at 37 8C in Dulbecco’s minimum essential medium

(DMEM) supplemented with 10% fetal calf serum,

streptomycin (100 mg/ml) and penicillin (100 U/ml) in a

humidiﬁed 5% CO

atmosphere. Host cell invasion assays

were carried out as detailed elsewhere (Yoshida et al.,

1989), by seeding the parasites onto each well of 24-well

plates containing 13-mm diameter round glass coverslips

coated with 1.5 £ 10

HeLa cells. After varying periods of

time, depending on the experiment, the duplicate coverslips

were washed in PBS and stained with Giemsa. The number

of intracellular parasites was counted in at least 500 Giemsa

stained cells.

2.2. Oral infection of mice with T. cruzi

Four-week-old female Balb/c mice were inoculated

orally with puriﬁed metacyclic trypomastigotes. The

parasites were introduced by intrapharyngeal route through

a plastic tube adapted to a 1 ml plastic syringe. Starting on

day 13 p.i., parasitemia was monitored by examining 5 ml

peripheral blood samples under phase contrast microscope,

twice a week.

2.3. Flow cytometry

Live metacyclic trypomastigotes (4 £ 10

) were incu-

bated for 1 h on ice, with monoclonal antibodies (MAbs)

directed to different T. cruzi surface molecules, or with

unrelated MAb 1C3 directed to Leishmania amazonensis

gp63 (Barbie

ri et al., 1993). After washings in PBS, the

parasites were ﬁxed with 2% paraformaldehyde in PBS for

30 min. The ﬁxative was washed out, and the parasites were

incubated with ﬂuorescein-labeled goat anti-mouse IgG for

1 h at room temperature. Following two more washes, the

number of ﬂuorescent parasites was estimated with a Becton

Dickinson FACscan cytometer. Assays with ﬁxed and

permeabilised parasites were carried out as follows: ﬁxation

with 2% paraformaldehyde, washings in PBS, treatment

with 0.1% saponin in PBS at room temperature for 30 min,

washings in PBS, incubation with MAb 3F6 for 1 h at room

temperature, washes in PBS, and incubation with ﬂuor-

escein-conjugated antibody as described above.

2.4. TEM and immunocytochemistry

Puriﬁed metacyclic trypomastigotes (5 £ 10

)were

washed in PBS and ﬁxed with a solution containing 0.1%

glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium

cacodylate buffer, pH 7.2. After 1 h at room temperature and

washings in cacodylate buffer, the parasites were treated

with PBS containing 0.1 M glycine, for 30 min at room

temperature, to block free aldehyde groups and then washed

in PBS. The parasites were blocked with PBS containing 5%

BSA for 1 h at room temperature and incubated with MAb

3F6 or with unrelated antibody, diluted 1:10 in blocking

solution, for 2 h at room temperature. Following washings

in PBS containing 1% BSA, the parasites were incubated for

2 h with anti-mouse IgG coupled to 10 nm colloidal gold

particles diluted 1:40 in blocking solution. After inclusion in

2% agarose and treatment with 1% OsO

for 1 h,

dehydration in ethanol series, the parasites were embedded

in Epon 812 (EMS). Thin sections were collected on

Formvar/carbon-coated copper grids, stained with 2%

uranyl acetate for 8 min and 1% lead citrate for 4 min,

V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860852

washed, dried and observed in transmission electron

microscope (Jeol 1200 EX II).

2.5. Detection of proteinase and trans-sialidase activities

T. cruzi proteinase activity was determined as follows:

3 £ 10

metacyclic trypomastigotes were lysed in 0.4%

Triton X-100, solubilised in sample buffer without 2-

mercaptoethanol, and then loaded onto 10% SDS-poly-

acrylamide gel containing 0.1% copolymerised gelatin as

substrate. After electrophoresis, the gels were subjected to

two 30 min washes with 2.5% Triton X-100 in 0.1 M acetate

buffer, pH 6.0, to remove SDS, and incubated overnight in

the same buffer without detergent. The gel was stained with

Coomassie blue R250 and destained for visualisation of

white bands against a blue background. Trans-sialidase

activity was determined as previously described (Schenk-

man et al., 1992). Brieﬂy, 10

metacyclic forms were

pelleted, detergent lysed and processed for measurement of

trans-sialidase activity, by the transfer of sialic acid from

sialyllactose to

D-glucose-1-[

C]lactose and detection of

sialylated products by chromatography in QAE-Sephadex

A-25 (Pharmacia).

2.6. Treatment of parasites with neuraminidase and drugs

that interfere with cell signaling

Metacyclic trypomastigotes were treated at 37 8C for 1 h

with 0.2 U/ml of neuraminidase (type III from Vibrio

cholerae or type X from Clostridium perfringens, Sigma) in

PBS, pH 6.0, containing 1 mM CaCl

. After washings in

PBS, the parasites were used for reactivity towards lectin

and for cell invasion assays. Treatment of metacyclic forms

with different drugs included incubation with 250 mM

genistein at 37 8C for 30 min, 1 mM U73122 at 37 8C for

4 min; 1 mM ionomycin plus 20 mM NH

Cl for 10 min at

37 8C.

2.7. Southern blot analysis

T. cruzi DNA was digested with different restriction

enzymes, separated by electrophoresis on 0.8% agarose gel

and blotted onto nylon membranes. Hybridisation with the

probe, which consisted of a DNA fragment corresponding to

ORF of gp82 gene (whole insert of gp82 cDNA clone)

labeled with [

P], and washings were performed as detailed

(Araya et al., 1994).

2.8. Pulsed ﬁeld gel electrophoresis

Agarose blocks containing genomic DNA from 10

parasites were prepared, incubated at 50 8C for 16 h in lysis

solution containing 10 mM Tris – HCl, pH 8.0, 500 mM

EDTA, 1% sarkosyl, 1 mg/ml proteinase K, equilibrated in

TE, washed and stored in 0.5 M EDTA at 4 8C. Small

portions (equivalent to 10

parasites) were electrophoresed

(1.2% agarose gel in 0.5 £ TBE) at 80 V for 132 h in Gene

Navigatore System (Pharmacia), from pulse times varying

from 90 to 800 s. DNA from Hansenula wingei was used as

reference. After transfer to nylon membranes, chromosomal

DNA bands were hybridised with the [

P]-labeled insert of

gp82 cDNA clone and revealed by exposure to X-ray ﬁlm

(Hyperﬁlm-MP, Amersham).

2.9. Statistics

Student’s t-test was used to determine signiﬁcance in T.

cruzi cell invasion assays, in which the infectivity between

CL isolate and clone CL-14 was compared or the effect of

treatment of parasites with neuraminidase or drugs that

inhibit signal transduction was evaluated.

3. Results

3.1. In vivo and in vitro infection by metacyclic forms

of T. cruzi isolate CL and clone CL-14

We compared the infectivity of CL isolate and clone

CL-14 by oral administration, a highly efﬁcient route

leading to systemic T. cruzi infection (Hoft, 1996; Neira

et al., 2003). Balb/c mice were injected orally with

2 £ 10

metacyclic forms of clone CL-14 or 4 £ 10

parasites of CL isolate. Starting on day 13 p.i., blood

samples were examined twice a week. Parasitemia was

not detectable in mice inoculated with clone CL-14

whereas the animals inoculated with ﬁve-fold fewer

metacyclic forms of CL isolate developed high para-

sitemias (Fig. 1A). The parasites used in these exper-

iments were . 95% pure typical metacyclic

trypomastigotes, with the kinetoplast posterior to the

nucleus, as visualised in Giemsa-stained preparations

(Fig. 2). We have noted that CL isolate metacyclic forms

were long and slender, with the kinetoplast localised

closer to the nucleus than to the posterior end, whereas

clone CL-14 parasites were shorter, with the kinetoplast

very close to the posterior end.

To examine whether the low infectivity of clone CL-

14 was associated with the impaired ability to invade

host cells, we performed invasion assays by incubating

metacyclic forms with HeLa cells for 1 h at 37 8C, at

parasite:cell ratio of 10:1. The number of internalised

parasites of clone CL-14 was approximately four-fold

lower as compared to that of the CL isolate (Fig. 1B), a

difference extremely signiﬁcant ðP , 0:0001Þ: Exper-

iments were also performed to determine the ability of

clone CL-14 to replicate intracellularly. Parasites were

incubated with HeLa cells for 1 h and, after washings in

PBS, one set of duplicate coverslips were immediately

stained with Giemsa whereas other sets were reconsti-

tuted with DMEM containing 2% fetal calf serum

V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860 853

and maintained for up to 72 h. No apparent intracellular

replication of clone CL-14 was observed by 72 h p.i., as

opposed to the CL isolate (Fig. 1C) that multiplied

actively as amastigotes.

3.2. Differential expression of gp82 on the surface of CL

isolate and clone CL-14

In CL isolate, gp82 is the main surface molecule of

metacyclic forms associated with the parasite infectivity

(Ramirez et al., 1993; Neira et al., 2003). We examined

whether clone CL-14 differed from CL isolate as regards

the expression of gp82. Analysis of metacyclic forms by

ﬂow cytometry, upon reaction of live parasites with MAb

3F6, consistently revealed in CL isolate a major

population expressing high gp82 levels and a minor

population with lower ﬂuorescence intensity, whereas in

clone CL-14 the parasites reacting poorly with MAb 3F6

predominated over those with higher gp82 expression

(Fig. 3A). We conﬁrmed the reduced expression of gp82

Fig. 1. Differential infectivity of metacyclic forms of Trypanosoma

cruzi CL isolate and clone CL-14. (A) The course of infection in mice

inoculated with parasites by oral route was monitored by counting the

number of parasites in 5 ml blood samples. Each data point corresponds

to the mean parasitemia of ﬁve animals in each group of mice. Note the

high parasitemias produced by CL isolate in contrast to the lack of

patent infection by clone CL-14. (B) Host cell invasion was assayed by

incubating parasites with HeLa cells at 37 8C for 1 h. After washings in

PBS, the number of intracellular parasites was counted in a total of at

least 500 Giemsa-stained cells. The difference in infectivity between CL

isolate and clone CL-14 was extremely signiﬁcant ðP , 0:0001Þ by

Student’s t-test. (C) Intracellular T. cruzi replication was examined by

incubating parasites with HeLa cells for 1 h. After washings in PBS,

one set of coverslips were stained with Giemsa whereas another set was

maintained for 72 h in Dulbecco’s minimum essential medium contain-

ing 2% FCS. Values are the means ^ SD of eight experiments (B) and

three experiments (C), performed in duplicate for each experiment.

Fig. 2. Metacyclic trypomastigotes of Trypanosoma cruzi CL isolate and

clone CL-14. Puriﬁed parasites were stained with Giemsa. Note the position

of kinetoplast very close to the posterior end in clone CL-14 and more

proximal to the nucleus in CL isolate.

Fig. 3. Expression of surface glycoproteins gp82 in metacyclic forms of

Trypanosoma cruzi CL isolate and clone CL-14. (A) Live parasites were

reacted with MAb 3F6 directed to gp82, ﬁxed and then processed as

described in Section 2 for analysis by ﬂow cytometry. Representative

results of at least seven experiments are shown. Note the lower ﬂuorescence

of clone CL-14, reﬂex of reduced gp82 expression. (B) Detergent extracts,

equivalent to 3 £ 10

parasites, were electrophoresed in 10% SDS-PAGE

gels and analysed by immunoblotting using MAb 3F6. Gp82 bands of

comparable intensity were revealed in CL isolate and clone CL-14. (C)

Parasites were permeabilised or not by treatment with saponin before

reaction with MAb 3F6 and processing for analysis by ﬂow cytometry.

Representative results of three experiments are shown. Note the increased

gp82 levels in permeabilised clone CL-14 parasites.

V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860854

on the surface of clone CL-14 metacyclic forms by

immunocytochemical reactions visualised at the electron

microscope (Fig. 4). In CL isolate, the presence of gp82

was revealed by MAb 3F6-colloidal gold complex

throughout the parasite membrane, including the ﬂagel-

lum, whereas in clone CL-14 metacyclic forms

the labeling was sparse. However, metacyclic forms of

CL isolate and clone CL-14 displayed gp82 bands of

comparable intensity when analysed by immunoblotting

(Fig. 3B), a method that uses extracts of detergent lysed

parasites and therefore can also detect internally localised

gp82, not accessible to MAb 3F6 in live CL-14

Fig. 4. Distribution of surface molecule gp82 in metacyclic forms of Trypanosoma cruzi CL isolate and clone CL-14, revealed by immunogold. Parasites were

incubated sequentially with MAb 3F6 directed to gp82 and anti-mouse IgG coupled to colloidal gold particles and then processed for electron microscopy. Note

the labeling of gp82 in CL isolate metacyclic forms (arrows). K, kinetoplast. Bar, 0.5 mm. Insets show the position of kinetoplast relative to the nucleus (N) and

to the posterior end of the parasite. Bar, 1 mm.

V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860 855

metacyclic forms. Accordingly, permeabilisation of CL-

14 parasites with saponin before reaction with MAb 3F6

increased the gp82 levels detectable by ﬂow cytometry,

whereas only a minor alteration in the proﬁle of gp82

expression was observed in permeabilised CL isolate

metacyclic forms as compared to the controls (Fig. 3C).

We also compared the genomic organisation of gp82

gene family in CL isolate and clone CL-14. When

hybridised with the insert of gp82 cDNA clone, Southern

blots of genomic DNA digested with restriction enzyme

Bam HI, Eco RI, Hind III or Hae III, displayed similar

proﬁles in CL isolate and clone CL-14 (Fig. 5A).

Chromosomal mapping of gp82 genes, carried out by

hybridising the same probe with chromosomal size

fragments separated by pulsed ﬁeld gel electrophoresis,

revealed some differences. A weak band of , 3.3 Mb and

a , 2.1 Mb fragment of high intensity were detected only

in CL isolate, whereas clone CL-14 displayed

an exclusive fragment of , 2.9 Mb and a , 1.7 Mb

band of much higher intensity than that of CL isolate

(Fig. 5B).

3.3. Expression of mucin-like surface molecule gp35/50

and of gp90 in CL isolate and clone CL-14

We also examined the proﬁle of other metacyclic

trypomastigote surface molecules that are known to interact

with host cells. By ﬂow cytometry, CL isolate and clone CL-

14 parasites were found to express on the surface

comparable levels of the mucin-like glycoprotein gp35/50

identiﬁed by MAb 2B10 (Fig. 6A) and immunoblots

revealed gp35/50 bands of similar intensity but displaying

some difference in size (Fig. 6B). The variant form of gp35/

50 as well as gp90, the stage-speciﬁc glycoprotein that

negatively modulates cell invasion (Ma

laga and Yoshida,

2001), recognised, respectively, by MAb 10D8 and MAb

1G7 in a number of poorly infective T. cruzi isolates (Ruiz

et al., 1998), were not detected in CL isolate or clone CL-14,

but both expressed a variant gp90 detectable by MAb 5E7

Fig. 5. Genomic organisation of gp82 genes in Trypanosoma cruzi CL

isolate and clone CL-14. (A) Southern blot of genomic DNA digested with

the indicated restriction enzyme was hybridised with the whole insert of

gp82 cDNA clone. Samples 1, 2, 3 and 4 correspond to CL isolate and l

and 4

to clone CL-14. (B) Chromosomal bands of parasites were

separated by pulsed ﬁeld gel electrophoresis, transferred to nylon

membrane and hybridised with the labeled insert of gp82 cDNA clone.

Numbers correspond to molecular sizes. Arrows point to fragments

detectable only in CL isolate and arrowheads to bands exclusive for or

that appear in higher intensity in clone CL-14.

Fig. 6. Molecular proﬁle of metacyclic forms of Trypanosoma cruzi CL

isolate and clone CL-14. (A) Live parasites were reacted with MAb 2B10

directed to gp35/50, ﬁxed and processed for analysis by ﬂow cytometry.

Representative results of seven experiments are shown. The comparable

ﬂuorescence intensity indicates that there is no difference in gp35/50

expression. (B) Detergent extracts, equivalent to 3 £ 10

parasites, were

electrophoresed in 10% SDS-PAGE gels and analysed by immunoblotting

with the indicated monoclonal antibodies. (C) For detection of proteases,

Triton X-100 extracts, equivalent to 3 £ 10

parasites, pre-incubated or not

with 100 mM E-64, were electrophoresed in 10% SDS-PAGE gel

containing gelatin. Afterwards, the gel was washed, incubated overnight

and stained with Coomassie blue. Note the high expression of cysteine

protease activity in clone CL-14.

V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860856

(Fig. 6B) directed to an epitope that is cryptic in live

parasites (Teixeira and Yoshida, 1986).

3.4. Cruzipain and trans-sialidase activities in CL isolate

and clone CL-14

Cruzipain, the major T. cruzi cysteine proteinase

(gp57/51) has been implicated in host cell invasion of

tissue culture trypomastigotes as well as in the replication of

amastigotes (Meirelles et al., 1992). We determined the

cruzipain activity of CL isolate and clone CL-14 metacyclic

forms in gelatin gels, by loading parasite preparations in

absence or in the presence of 20 mM E-64, an inhibitor of

cysteine proteinase. Expression of functional cruzipain,

inhibitable by E-64, was higher in clone CL-14 (Fig. 6C). In

addition to the gp57/51 bands, a minor band migrating

slower than BSA and resistant to E-64 was detected in clone

CL-14. As regards trans-sialidase, also implicated in cell

invasion of tissue culture trypomastigotes (Schenkman et al.,

1991), the activity in clone CL-14 was , 1.6-fold higher

than in CL isolate.

3.5. Differential effect of neuraminidase treatment of CL

isolate and clone CL-14 metacyclic forms on host cell

invasion

A previous study had shown that, depending on the T.

cruzi isolate, treatment of metacyclic trypomastigotes with

neuraminidase, which removes sialic acid from mucin-like

molecule gp35/50, increases the ability to invade target cells

(Yoshida et al., 1997). To determine whether removal of

sialic acid affected the infectivity of clone CL-14 metacyclic

forms, the parasites were treated with neuraminidase as

described in Section 2. Enzyme-treated parasites had

augmented their reactivity with Bauhinia purpurea lectin,

which has afﬁnity for

D-galactose, an indication that sialic

acid was removed, exposing galactosyl residues. As shown

in Fig. 7A, the infectivity of clone CL-14 metacyclic forms

Fig. 7. Differential effect of treatment of Trypanosoma cruzi metacyclic forms with different drugs on target cell invasion of CL isolate and clone CL-14. (A)

Parasites were either untreated or treated with neuraminidase (0.2 U/ml) for 1 h, washed and then seeded onto HeLa cells. After 1 h incubation at 37 8C,

followed by washings in PBS, the number of intracellular parasites was counted in a total of at least 500 Giemsa-stained cells. Values are the means ^ SD of

four experiments performed in duplicate. The difference between untreated controls and neuraminidase-treated CL-14 parasites was signiﬁcant, whether the

neuraminidase used was from Vibrio cholerae ðP , 0:05Þ or from Clostridium perfringens ðP , 0:005Þ; by Student’s t-test. (B) Parasites, pre-treated or not

with the indicated drug, were incubated with HeLa cells, at parasite:cell ratio of 50:1 for clone CL-14 and 10:1 for CL isolate. After 30 min at 37 8C, the number

of intracellular parasites was counted in at least 500 Giemsa-stained cells. Values are the means ^ SD of three experiments performed in duplicated. For CL

isolate, a signiﬁcant inhibition of cell invasion was observed upon treatment of parasites with genistein ðP , 0:0001Þ or U73122 ðP , 0:0001Þ; whereas for

clone CL-14 the difference between untreated controls and parasites treated with the combination ionomycin þ NH

Cl was signiﬁcant ðP , 0:05Þ; by

Student’s t-test. This suggests phospholipase C-mediated Ca

2þ

release in CL isolate and phospholipase C-independent Ca

2þ

mobilisation from

acidocalcisomes in clone CL-14.

V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860 857

towards HeLa cells increased signiﬁcantly upon treatment

with neuraminidase from Vibrio cholerae ðP , 0:05Þ or

Clostridium perfringens ðP , 0:005Þ; whereas that of CL

isolate remained unaltered. Neuraminidase-treated clone

CL-14 parasites failed to replicate intracellularly.

3.6. Activation of distinct signaling pathways in metacyclic

forms of CL isolate and clone CL-14 during host cell

invasion

In this set of short time (30 min) cell invasion assays, in

which parasites were pre-treated with drugs that act on

signal transduction pathways leading to Ca

2þ

mobilisation,

the parasite:cell ratio used for clone CL-14 was 50:1.

Genistein, a speciﬁc inhibitor of protein tyrosine kinase

(Akiyama et al., 1987), that inhibits Ca

2þ

response and the

infectivity of CL isolate metacyclic forms (Yoshida et al.,

2000), did not affect HeLa cell entry of clone CL-14

parasites (Fig. 6B). Treatment with U73122, a speciﬁc

inhibitor of phospholipase C (Bleasdale et al., 1990), which

mediates inositol 1,4,5-triphosphate production (Berridge,

1993), inhibited HeLa cell invasion of CL isolate but not of

clone CL-14 metacyclic forms, whereas Ca

2þ

ionophore

ionomycin plus NH

Cl, a combination that potentiates Ca

2þ

release from acidocalcisomes (Docampo et al., 1995), had

an opposite effect, by affecting only clone CL-14 internal-

isation (Fig. 7B).

4. Discussion

Our study supports the hypothesis that the metacyclic

stage-speciﬁc surface glycoprotein gp82 is a key molecule in

promoting efﬁcient T. cruzi infection. Of the various parasite

molecules implicated in host cell invasion analysed in this

study, gp82 was found to be expressed at much lower levels on

thesurface ofclone CL-14, ascompared tothehighly infective

parental CL isolate, but otherwise the molecular proﬁles of the

isolate and the clone were similar. In CL isolate, gp82 plays a

central role in target cell penetration in vitro (Ramirez et al.,

1993; Ruiz et al., 1998) as well as in promoting mucosal

infection, leading to high parasitemias, upon oral challenge

(Neira et al., 2003). The expression of reduced levels of

surface gp82 in metacyclic froms of clone CL-14 could

explain the poor cell invasive capacity of these parasites in

vitro and why they do not produce patent infection in mice

when administered orally. Gp82, which binds to gastric mucin

in a dose-dependent manner (Neira et al., 2003), may ﬁrst

adhere to mucin that lines the mucosal surfaces, in order to

initiate mucosal infection. If engagement of gp82 is required

to adhere to and traverse the mucin coat, before parasite

penetration into underlying epithelial cells, the reduced

expression of gp82 in CL-14 metacyclic forms would be an

impediment for this initial interaction with the host.

Based on several pieces of evidence, we had proposed

that the most efﬁcient target cell penetration of metacyclic

trypomastigotes is mediated by gp82, which triggers in both

cells the activation of signaling cascades leading to Ca

2þ

mobilisation from intracellular reservoirs (Ruiz et al., 1998;

Neira et al., 2003). Binding of gp82 to its receptor on the

host cell induces in CL isolate metacyclic forms the

activation of protein tyrosine kinase and Ca

2þ

release,

probably from endoplasmic reticulum, in a manner

dependent on inositol 1,4,5-triphosphate (Yoshida et al.,

2000). In accord with this, cell invasion of clone CL-14 that

is deﬁcient in the expression of surface gp82 is not

dependent on protein tyrosine kinase. An alternative signal

transduction pathway must be induced in metacyclic forms

of clone CL-14 during target cell entry in vitro. One

possibility is that the mucin-like surface glycoprotein gp35/

50 is involved.

Gp35/50, which also induce target cell Ca

2þ

response,

although to a lesser degree than gp82 (Dorta et al., 1995),

has been implicated in host cell invasion of metacyclic

forms of poorly infective T. cruzi isolates (Ruiz et al., 1998).

This molecule is the main acceptor of sialic acid in a

reaction mediated by trans-sialidase (Schenkman et al.,

1993), but sialic acid appears to impair, rather than promote,

the interaction of gp35/50 with target cells. Treatment of

metacyclic forms with neuraminidase removes sialic acid

from gp35/50, increases the Ca

2þ

signal-inducing activity

and, depending on the T. cruzi isolate, augments the parasite

infectivity (Yoshida et al., 1997). Neuraminidase treatment

of CL-14 metacyclic forms increased the reactivity with

lectin recognising

D-galactose as well as the invasive

capacity of clone CL-14 metacyclic forms towards HeLa

cells, suggesting that the removal of sialic acid from gp35/

50 facilitates parasite– target cell interaction by exposing

D-

galactose residues, which putatively are required for

recognition of target cell receptors. Neuraminidase treat-

ment did not affect CL isolate infectivity, which is not

dependent on gp35/50.

Our results indicate that distinct signaling cascades are

activated in CL isolate and clone CL-14 parasites during

host cell invasion, leading to Ca

2þ

mobilisation from

different intracellular reservoirs. Ca

2þ

release in CL

isolate, possibly from endoplasmic reticulum (Neira et al.,

2002) is mediated by inositol 1,4,5-triphosphate gener-

ated by phospholipase C, an enzyme whose inhibition

results in decreased parasite infectivity. In clone CL-14,

the Ca

2þ

necessary for parasite internalisation appears,

at least in part, to be mobilised from acidocalcisomes in

a manner independent of inositol 1,4,5-triphosphate

(Table 1).

In addition to a reduced ability to enter host cells, clone

CL-14 also appears to have impaired capacity for intra-

cellular development (Fig. 1C). As T. cruzi cysteine

proteinase cruzipain plays a role in intracellular parasite

replication (Meirelles et al., 1992), one possibility could be

a deﬁcient replication due to the reduced cruzipain activity.

However, this is apparently not the case. Expression of

functional cruzipain was higher in clone CL-14 as compared

V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860858

to CL isolate. In addition, Paiva et al. (1998) have found

similar expression of cruzipain on ﬂagellar and cellular

membranes of trypomastigotes of CL isolate and clone CL-

14. Another possibility could be an impaired capacity to

escape from the parasitophorous vacuole to the cytoplasm,

due to a deﬁcient expression of trans-sialidase, an enzyme

that also displays neuraminidase activity (Schenkman et al.,

1992). According to Hall et al. (1992), T. cruzi neurami-

nidase facilitates the parasite’s escape into the cytoplasm by

desialylating the vacuole membrane and rendering it more

susceptible to lysis by a parasite hemolysin. That possibility

is not compatible with trans-sialidase activity of clone CL-

14, which was , 1.6-fold higher than that of the CL isolate.

An intriguing question is whether the deﬁcient expression in

clone CL-14 of gp82, which is a member of T. cruzi gp85/

sialidase family (Araya et al., 1994) but is apparently devoid

of enzymatic activity, impairs functions essential for

parasite development other than the ability to bind to

gastric mucin and to enter epithelial cells.

Taken together, our data suggest that the non-virulence

of clone CL-14 metacyclic forms is associated with the

reduced expression on the surface of gp82, the molecule

involved in mucosal infection in vivo and activation of

parasite signaling pathway leading to efﬁcient host cell

invasion.

Acknowledgements

This work was supported by Fundac¸a

o de Amparo a

Pesquisa do Estado de Sa

o Paulo (FAPESP). We thank

Evania B. Azevedo for help in the detection of parasite

trans-sialidase activity, and Dr Tania A.T. Gomes for

reading the manuscript.

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Table 1

Activation of distinct signal transduction pathways in metacyclic forms of

Trypanosoma cruzi CL isolate and clone CL-14 during host cell invasion

Parasite component

involved

Isolate CL Clone CL-14

Surface glycoprotein gp82 gp35/50

Protein tyrosine kinase Yes No

Phospholipase C Yes No

Intracellular Ca

2þ

store Inositol 1,4,5-triphosphate-

sensitive compartment

Acidocalcisome

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V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860860

INFECTION AND IMMUNITY, July 2007, p. 3264–3270 Vol. 75, No. 7

0019-9567/07/$08.00ϩ0 doi:10.1128/IAI.00262-07

Expression and Cellular Localization of Molecules of the gp82 Family

in Trypanosoma cruzi Metacyclic Trypomastigotes

ᰔ

Vanessa D. Atayde, Mauro Cortez, Renata Souza, Jose´ Franco da Silveira, and Nobuko Yoshida*

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo,

Rua Botucatu 862-6° andar, 04023-062, Sa˜o Paulo, Brazil

Received 16 February 2007/Returned for modiﬁcation 22 March 2007/Accepted 6 April 2007

A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and

identiﬁed by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82

deﬁned by MAb 3F6, no information is available on members of this protein family. From cDNA clones

encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant proteins J18 and C03,

the former containing and the latter lacking the epitope for MAb 3F6. Polyclonal antibodies to J18 and C03

proteins were generated and used, along with MAb 3F6, to analyze the expression and cellular localization of

gp82 family members in metacyclic forms of CL and G strains, which belong to highly divergent genetic groups.

By two-dimensional gel electrophoresis and immunoblotting, molecules of 82 to 86 kDa, focusing at pH 4.6 to

5.4, and molecules of 72 to 88 kDa, focusing at pH 4.9 to 5.7, were visualized in CL and G strains, respectively.

Flow cytometry and microscopic analysis revealed in both strains similar expression of MAb 3F6-reactive gp82

in live and permeabilized parasites, indicating its surface localization. The reaction of live parasites of both

strains with anti-J18 antibodies was weaker than with MAb 3F6 and was increased by permeabilization.

Anti-C03 antibodies bound predominantly to ﬂagellar components in permeabilized G strain parasites, but in

the CL strain the ﬂagellum was not the preferential target for these antibodies. Host cell invasion of metacyclic

forms was inhibited by J18 protein, as well as by MAb 3F6 and anti-J18 antibodies, but not by C03 protein or

anti-C03 antibodies.

Metacyclic trypomastigotes of different Trypanosoma cruzi

strains may engage different surface molecules to invade host

cells (32). In the highly infective CL strain, the metacyclic

stage-speciﬁc surface glycoprotein gp82 identiﬁed by monoclo-

nal antibody (MAb) 3F6 promotes target cell invasion by trig-

gering bidirectional signaling cascades leading to Ca

2ϩ

mobi-

lization in both parasite and target cells (17, 22, 34), which is an

event essential for parasite internalization (12, 26). Binding of

gp82 to target cells induces a Ca

2ϩ

-dependent disruption of

actin microﬁlaments (10), a process reported to facilitate par-

asite entry (21). gp82 also has the ability to bind to gastric

mucin (18), and this is crucial for the establishment of T. cruzi

infection by the oral route since the binding to mucin repre-

sents the ﬁrst step toward the invasion of gastric mucosal

epithelium (8, 9). The poorly invasive G strain metacyclic

forms express MAb 3F6-reactive gp82 molecules, but they

preferentially use the mucin-like surface glycoproteins gp35/50

to enter host cells (22, 24, 33).

The MAb 3F6-reactive gp82 molecule is a member of a

multigene family, which is part of the large trans-sialidade/gp85

gene family (1). According to T. cruzi proteome analysis, 30 of

the 50 top-scoring proteins detected exclusively in the infective

trypomastigote forms are trans-sialidase (TS) family members

(2). The repertoire of metacyclic trypomastigote gp82 mole-

cules, the degree to which they are heterogeneous, the expres-

sion of members lacking the MAb 3F6 epitope, and their

presence in locations other than on the parasite surface are

matters that remain to be determined. The gp82 molecules

characterized so far are highly conserved in T. cruzi strains CL

and G, which belong to highly divergent genetic groups (5),

and show 97.9% peptide sequence identity overall and 100%

identity with regard to the cell binding site and the epitope for

MAb 3F6 (32).

In this study we isolated and characterized a new member of

the gp82 family and performed a global analysis on the expres-

sion as well as the cellular localization of gp82 proteins in

metacyclic forms of the CL and G strains. The strategy con-

sisted of the following steps: (i) isolation of a cDNA clone

encoding a member of the gp82 family lacking the epitope for

MAb 3F6, (ii) production of recombinant proteins with and

without the MAb 3F6 epitope, (iii) generation of antibodies

against the referred recombinant proteins, and (iv) two-dimen-

sional (2D) gel electrophoresis of metacyclic trypomastigote

extracts and immunoblotting in parallel with analysis by ﬂow

cytometry and ﬂuorescent microscope visualization of live as

well as permeabilized parasites, using MAb 3F6 and anti-gp82

polyclonal antibodies.

MATERIALS AND METHODS

Parasites. The following T. cruzi strains were used: CL, isolated from the

insect Triatoma infestans in the state of Rio Grande do Sul (4), and G, isolated

from an opossum in the Amazon (31). Parasites were maintained cyclically in

mice and in liver infusion tryptose. Before puriﬁcation, in some cases the para-

sites were grown in Grace’s medium. Metacyclic forms from cultures in liver

infusion tryptose or Grace’s medium at the stationary growth phase were puriﬁed

by passage through a DEAE-cellulose column, as described previously (27).

Puriﬁcation of RNA, RT-PCR, and cloning in pGEM-T. Puriﬁed CL strain

metacyclic trypomastigotes (1 ϫ 10

) were lysed with 1 ml of Trizol reagent

(Invitrogen). Following complete dissolution and the addition of 0.2 ml of chlo-

* Corresponding author. Mailing address: Escola Paulista de Medi-

cina, Universidade Federal de Sa˜o Paulo, Rua Botucatu 862-6° andar,

04023-062 Sa˜o Paulo, S.P., Brazil. Phone: 55 11 5576 4532. Fax: 55 11

5571 1095. E-mail: [email protected].

ᰔ

Published ahead of print on 16 April 2007.

3264

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roform, the parasite preparation was centrifuged at 14,000 ϫ g for 15 min at 4°C.

The aqueous phase was collected, and an equal volume of isopropyl alcohol was

added to precipitate the total RNA. After washing with 75% ethanol, the pre-

cipitate was resuspended in RNase-free water. Reverse transcription-PCR (RT-

PCR) was performed using the Access Quick RT-PCR system (Promega). The

reaction mixture contained the following: total T. cruzi RNA; deoxynucleoside

triphosphate mix; avian myeloblastosis virus reverse transcriptase; Taq polymer-

ase; the forward primer SL (5Ј-GATACAGTTTCTGTACTATATTGAG-3Ј),

which is speciﬁc for the spliced leader sequence (GenBank accession no.

M30787); and the reverse primer 1775R (5Ј-GTTCCATTCGAAAGCATCCA

GTT-3Ј), which is speciﬁc for a sequence of the 3Ј region of the gp82 gene

(GenBank accession no. L14824). Production of cDNA was carried out at 48°C

for 45 min. Ampliﬁcation was performed by running 45 cycles of denaturing,

annealing, and elongation at 94°C for 15 s, 44°C for 30 s, and 72°C for 1 min,

respectively. After puriﬁcation with a GenClean kit (Bio 101), the PCR product

was cloned in the plasmid vector pGEM using a pGEM-T Easy Vector kit

(Promega). Following ligation to the vector, the product was transformed in

Escherichia coli strain DH5␣, and the colonies were grown in LB broth. Inserts

released from cDNA clones were screened with a

P-labeled probe containing

the full-length T. cruzi gp82 gene. The selected clones were sequenced using a Big

Dye terminator cycle sequencing ready reaction kit (Perkin-Elmer).

Production and puriﬁcation of recombinant proteins J18 and C03. The re-

combinant protein J18, containing the full-length T. cruzi gp82 in frame with

glutathione S-transferase, was produced in E. coli DH5␣ by transforming the

bacteria with a pGEX-3 construct comprising the gp82 gene (23). All steps for

induction of the recombinant protein J18 and its puriﬁcation are detailed else-

where (10). The recombinant protein C03, containing the full-length gp82 in

fusion with six histidine residues, was produced in E. coli BL21(DE3) by trans-

forming the bacteria with the construct pHIS-C03. This construct was generated

by PCR using the following primers: one containing the third ATG initiation

codon plus an artiﬁcial BamHI site (5Ј-ATTGGATCCGATGTGCTGCGCCA

CC-3Ј) and the other containing the stop codon plus an artiﬁcial HindIII site

(5Ј-GGAAGCTTTCTCAGTAAAGGGCCGC-3Ј). As a template, we used the

plasmid pGEM-T-C03. Following cloning in the vector pET-22(bϩ) (pHIS;

Novagen, Madison, WI), the clones were digested with BamHI/HindIII in order

to release the insert. Upon conﬁrmation of the sequence, the clone pHIS-C03

was selected for further characterization. The transformed bacteria were grown

in LB medium and induced with 1 mM isopropyl-␤-

D-thiogalactopyranoside for

4 h at 37°C, treated with 15 mg of lysozyme in phosphate-buffered saline (PBS)

for 30 min at room temperature, and then sonicated for 20 min and centrifuged

at 12,000 ϫ g for 30 min a 4°C. After three washings with 10 ml of CHAPS

(3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) buffer at 1%,

the precipitate was collected, resuspended in binding buffer (20 mM Na

HPO

0.5 M NaCl, 20 mM imidazole, 8 mM urea, pH 7.4), and incubated at room

temperature under agitation for 2 h. Thereafter, the sample in binding buffer was

centrifuged at 2,600 ϫ g for 10 min, and the supernatant was added to a 5-ml

3ϩ

column (Ni Sepharose 6 Fast Flow; Amersham Biosciences). Following

incubation for 30 min under agitation at room temperature, the bound protein

containing the histidine tail was eluted with the elution buffer (20 mM Na

HPO

0.5 M NaCl, 250 mM imidazole, 8 M urea, pH 7.4) and dialyzed against double-

distilled water for 48 h at 4°C. The amount of puriﬁed protein was quantiﬁed by

reaction with Coomassie Plus (Pierce) in 96-well plates, and readings were taken

at 620 nm. To certify that the desired protein was obtained, the puriﬁed samples

were analyzed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) gels stained with Coomassie blue and by immunoblotting using

anti-His antibodies (Amersham Biosciences).

Generation of antibodies to recombinant proteins J18 and C03. BALB/c mice

were injected by intraperitoneal route with recombinant protein (5 ␮g/mouse) in

the presence of aluminum hydroxide (0.5 mg/mouse) as adjuvant. Fourteen days

later, all animals received the same amount of antigen plus adjuvant. Thereafter,

at 1-week intervals mice were given two more doses. Ten days after the last

immunizing dose, the mice were bled, and sera were collected and stored at

Ϫ20°C until used.

Isoelectric focusing, SDS-PAGE, and immunoblotting. The standard Western

blot analysis was performed, as previously described (33), by applying NP-40-

solubilized parasite extracts corresponding to 3 ϫ 10

cells into each well of 10%

SDS-PAGE gels. For 2D electrophoresis, samples containing 5 ϫ 10

metacyclic

trypomastigotes were washed three times in 25 mM HEPES, pH 7.4, containing

0.9% NaCl; samples were boiled for 5 min in 0.2% SDS lysis buffer and main-

tained on ice before incubation with 2D buffer (7 M urea, 2 M thiourea, 1%

dithiothreitol [DTT], 2% Triton X-100, 0.5% immobilized pH gradient buffer,

pH 4 to 7) containing protease inhibitors (100 ␮M phenylmethylsulfonyl ﬂuoride,

1 ␮M pepstatin, 100 ␮M leupeptin, and 5 mM EDTA) for 30 min at room

temperature. After centrifugation at 23,100 ϫ g for 10 min, the supernatant was

applied to immobilized pH gradient gel strips with 0.5% (pH 4.0 to 7.0) ampho-

lites (Amersham Biosciences). Isoelectric focusing was performed in an Ettan

IPGphor system (Amersham Biosciences) by applying 500 V, 1,000 V, 8,000 V,

and 7,400 V, sequentially at 1-h intervals. Thereafter, the gel strips were incu-

bated for 15 min in a solution containing 6 M urea, 50 mM Tris, pH 8.8, 30%

glycerol, 2% SDS, and 25 mM DTT. Following another 15-min incubation in this

solution without DTT but containing 125 mM iodoacetamide plus 0.02% bro-

mophenol blue, the samples were subjected to electrophoresis in 10% SDS-

PAGE gels. The proteins were then transferred to a nitrocellulose membrane,

which was processed for reaction with anti-gp82 antibodies.

Flow cytometry. Live metacyclic trypomastigotes (3 ϫ 10

) were incubated for

1 h on ice with anti-gp82 antibodies. After washings in PBS and ﬁxation with 2%

paraformaldehyde for 30 min, the parasites were incubated with anti-mouse

immunoglobulin G (IgG) conjugated to ﬂuorescein at room temperature for 1 h.

Following two more washes, the number of ﬂuorescent parasites was estimated

with a Becton Dickinson FACscan cytometer. Assays with ﬁxed and permeabil-

ized parasites were carried out as follows: ﬁxation with 2% paraformaldehyde for

30 min, washings in PBS, treatment with 0.1% saponin in PBS at room temper-

ature for 30 min, washings in PBS, and incubation with antibodies as described

above.

Microscopic visualization of ﬂuorescent parasites. Live metacyclic forms were

incubated for1honicewith anti-gp82 antibodies, washed, ﬁxed with 3.5%

formaldehyde in PBS, and placed onto glass slides and dried. Afterwards, the

parasites were incubated sequentially with ﬂuorescein-conjugated anti-mouse

IgG diluted 1:40 in PGN (0.15% gelatin in PBS containing 0.1% sodium azide)

for 1 h, and 10 ␮M DAPI (4Ј,6Ј-diamidino-2-phenylindole; Molecular Probes)

for visualization of kinetoplast and nucleus. Images were acquired on a Nikon

E600 ﬂuorescence microscope coupled to a Nikon DXM 1200F digital camera

using ACT-1 software. In parallel, the parasites were ﬁrst ﬁxed with 3.5% form-

aldehyde, washed, and then processed as above, except that a 1-h incubation with

anti-gp82 antibodies was carried out in the presence of 0.1% saponin in PGN for

parasite permeabilization.

Host cell invasion assay. HeLa cells, the human carcinoma-derived epithelial

cells, were grown at 37°C in Dulbecco’s minimum essential medium, supple-

mented with 10% fetal calf serum, streptomycin (100 ␮g/ml), and penicillin (100

U/ml) in a humidiﬁed 5% CO

atmosphere. Cell invasion assays were carried out

as detailed elsewhere (33) by seeding the parasites onto each well of 24-well

plates containing 13-mm-diameter round glass coverslips coated with 1.5 ϫ 10

HeLa cells. After a 1-h incubation with parasites at a parasite:cell ratio of 10:1,

the coverslips were washed in PBS and stained with Giemsa, and the numbers of

intracellular parasites were counted.

Nucleotide sequence accession number. The sequence of the recombinant

protein C03 has been deposited in the GenBank database under accession

number EF445668.

RESULTS

Isolation of a new member of the gp82 family. We cloned a

full-length gp82 cDNA (C03) by RT-PCR, using a set of prim-

ers based on the sequence of clone J18, which codes for a gp82

protein containing the epitope for MAb 3F6 (1) and the

miniexon sequence (29) present in all trypanosomatid mRNAs.

Translation of C03 cDNA in each of the six possible reading

frames indicated only one large open reading frame with three

in-frame ATG initiator codons. Within this open reading

frame the ﬁrst potential start codon is separated from the

spliced leader sequence by 89 bp. The second and third start

codons are in the same reading frame as the ﬁrst ATG and they

are 51 and 159 bp downstream from it, respectively. Analysis of

the 5Ј sequences ﬂanking the three potential start codons in-

dicates that the third ATG best ﬁts the Kozak eukaryotic

consensus sequences (15). This argues for the use of the third

ATG as the initiating methionine. However, we cannot exclude

the possibility of translation from the ﬁrst or the second in-

frame methionine, provided that the amino acid sequence of

the amino-terminal end of the native gp82 has not yet been

determined. Assuming the third codon as the initiating methi-

VOL. 75, 2007 EXPRESSION OF T. CRUZI gp82 FAMILY PROTEINS 3265

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onine, C03 cDNA encodes a polypeptide of 668 amino acids

with a predicted molecular mass of 72.54 kDa and pI 5.03 (Fig.

1). It has two highly conserved copies of the sialidase motif

SXDXGXTW (Asp box) and a complete copy of the subter-

minal motif VTVKNVFLYNR characteristic of all members of

the TS superfamily (13). Although C03 does not display an

N-terminal sequence predicted to be a signal peptide by the

SignalP 3.0 program (3), it has a typical glycosylphosphatidyl-

inositol anchor signal sequence at the carboxy terminus (Fig. 1).

The amino acid sequence deduced from cDNA clone C03

FIG. 1. Sequences of proteins of T. cruzi metacyclic trypomastigote gp82 family members. Shown are the proteins C03 (CL strain), R31 (CL

strain), and J18 (G strain). In proteins R31 and J18 are the sequences (indicated by lines above the sequences) identiﬁed as the epitope for MAb

3F6 (P3) and as the conformational cell binding site (P4 and P8). The alternative initiator methionines in the C03 are indicated by asterisks. The

conserved motif VTV, characteristic of all members of the gp85/TS family, and the potential glycosylphosphatidylinositol (GPI) anchor consensus

sequence are indicated (lines above sequences). Points represent residues that are conserved in the three proteins, nonconserved amino acids are

indicated, and dashes represent residues that are lacking in the indicated sequence. Note the high degree of identity between protein R31 and J18,

as opposed to the much lower sequence identity of these proteins in relation to protein C03.

3266 ATAYDE ET AL. I

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displayed considerable differences from the sequences de-

duced from clones J18 and R31 (GenBank accession no.

AF128843) isolated in previous studies from cDNA libraries

from G and CL strain metacyclic trypomastigotes, respectively.

The overall amino acid sequence identity was 59.1% between

C03 and J18 proteins and 49% between C03 and R31 proteins.

Comparative analysis of recombinant proteins C03 and J18.

Using synthetic peptides based on the J18 protein sequence,

the epitope for MAb 3F6 has been mapped, and it corresponds

to the sequence represented by peptide P3 (16). As shown in

Fig. 2A, compared to the sequence of J18 corresponding to P3,

the most conspicuous difference in the C03 protein was the

substitution of three uncharged residues by charged ones (Fig.

2A). Reﬂecting this difference, the C03 protein failed to be

recognized by MAb 3F6 (Fig. 2B). Considerable differences

were also seen in the sequence corresponding to the cell bind-

ing site, which in the J18 protein is formed by juxtaposition of

two sequences, P4 and P8, containing several charged residues

separated by a more hydrophobic stretch (16); comparison of

the sequences of the two proteins revealed substitutions of

charged residues for uncharged amino acids and vice versa

(Fig. 2A). Accordingly, the binding capacity of the C03 protein

to HeLa cells was lower than that of the J18 protein (Fig. 2C).

Expression and cellular localization of molecules of the

gp82 family in G and CL strain metacyclic forms. We gener-

ated antibodies against C03 and J18 recombinant proteins by

immunizing groups of mice with either of these proteins. The

anti-C03 antibody also reacted with J18 protein, and anti-J18

antibodies recognized C03 protein as well, and in both cases

the reaction was stronger with the protein that served as im-

munogen (Fig. 3). Both antibodies reacted similarly with gp82

molecules from metacyclic trypomastigotes of G and CL

strains (Fig. 3). Immunoblots of CL strain parasite extracts

subjected to 2D gel electrophoresis showed the MAb 3F6-

reactive gp82 molecules focusing at a pH range of 4.6 to 5.1

(data not shown). We conﬁrmed the presence of these gp82

molecules on the parasite surface by ﬂow cytometry analysis

and observation with ﬂuorescence microscopy, which revealed

no difference in the reactivity of live and permeabilized para-

sites with MAb 3F6 (Fig. 4). As regards the gp82 molecules

recognized by anti-J18 or anti-C03 antibodies, with isoelectric

points ranging from pH 4.6 to 5.4 (data not shown), their

location was at least in part intracellular, as deduced from the

high reactivity of permeabilized parasites compared to their

FIG. 2. Properties of recombinant proteins J18 and C03. (A) The

sequences identiﬁed as the epitope for MAb 3F6 (P3) and as the con-

formational cell binding site (P4 and P8) are aligned to show the

differences between J18 and C03 proteins, with asterisks indicating the

changed residues. (B) Immunoblot of puriﬁed J18 and C03 proteins

using MAb 3F6. Note the lack of reactivity of C03 protein. (C) Binding

of proteins J18 and C03 to HeLa cells, assayed as described in Mate-

rials and Methods. The reaction was revealed by sequential incubation

with polyclonal antiserum directed to J18 or to C03 and anti-mouse

IgG conjugated to peroxidase. OD

492

, optical density at 492 nm.

FIG. 3. Cross-reactivity between anti-J18 and anti-C03 antibodies.

Puriﬁed recombinant proteins J18 and C03, as well as the metacyclic

trypomastigote extract of T. cruzi strains G and CL, were analyzed by

immunoblotting using polyclonal antibodies to J18 or C03 protein.

FIG. 4. Cellular localization of proteins of the gp82 family in meta-

cyclic forms of the T. cruzi CL strain. Live or permeabilized parasites

were reacted with MAb 3F6 or polyclonal antibodies to J18 or C03 and

processed for analysis by ﬂow cytometry or for visualization by ﬂuo-

rescence microscopy. DAPI staining of parasite kinetoplast and nu-

cleus appears in blue. Note the similar expression patterns of MAb

3F6-reactive molecules in live and permeabilized parasites, whereas

the molecules recognized by anti-J18 and anti-C03 antibodies were in

part localized intracellularly, as indicated by the high ﬂuorescence inten-

sity of permeabilized parasites compared to their live counterparts.

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live counterparts, and this was clearly visible by observations

with ﬂuorescence microscopy (Fig. 4). We also noted that in

live parasites the distribution of molecules recognized by anti-

C03 antibodies was not homogeneous, particularly in the pos-

terior end, where they were more concentrated at some spots

(Fig. 4).

In G strain metacyclic forms, 2D gel electrophoresis and

immunoblotting revealed a similar proﬁle of gp82 bands focus-

ing at pH 4.9 to 5.7 when MAb 3F6 or anti-J18 antibodies were

used (data not shown). As in the CL strain, the MAb 3F6-

reactive gp82 molecules were on the surface, and no difference

in reactivity was observed between live and permeabilized par-

asites (Fig. 5). On the other hand, a portion of gp82 molecules

reacting with anti-J18 antibodies was intracellularly located, as

indicated by the considerably increased reactivity when the

parasites were permeabilized (Fig. 5). The most striking ob-

servation in the G strain was the reaction of anti-C03 antibod-

ies with permeabilized metacyclic forms. These antibodies,

which reacted with gp82 bands focusing at pH 4.9 to 5.5 (data

not shown), barely recognized live parasites but reacted in-

tensely with ﬂagellar components of permeabilized cells (Fig.

5), which means that these are intraﬂagellar molecules. In

order to determine whether the ﬂagellar location of proteins

recognized by anti-C03 antibodies was a characteristic of

strains of group 1 T. cruzi, to which the G strain belongs (5),

two other strains of this group were examined. The same pro-

ﬁle of the G strain, with the predominant reaction with ﬂagella,

was revealed in permeabilized metacyclic forms of these strains

(data not shown).

Effect of different proteins and antibodies on host cell inva-

sion by metacyclic trypomastigotes. To test the effect of pro-

teins and antibodies generated in this study, we used CL strain

metacyclic forms and epithelial HeLa cells. Parasites were in-

cubated with HeLa cells in the absence or presence of puriﬁed

recombinant protein, J18, C03, or glutathione S-transferase, at

40 ␮g/ml for 1 h. In contrast to the J18 protein, which inhibited

parasite internalization by ϳ65%, C03 protein had no inhibi-

tory effect (Fig. 6A). Why the C03 protein does not inhibit T.

cruzi invasion even though it binds to host cells, albeit to a

lesser extent than J18 (Fig. 2C), is not known. One possibility

is that the C03 protein binds to a target receptor distinct from

the J18 receptor, which recognizes a sequence different from

that corresponding to P4/P8. We also examined the effect of

anti-C03 and anti-J18 antibodies on HeLa cell invasion. Para-

sites were incubated for 30 min with anti-J18 or anti-C03 an-

tiserum diluted 1:10 or with ascitic ﬂuid containing MAb 3F6

diluted 1:10. After the antibody was washed out, the parasites

were seeded onto HeLa cells. As shown in Fig. 6B, parasite

entry was inhibited ϳ50% by MAb 3F6 and ϳ35% by anti-J18

antibodies, whereas anti-C03 antibodies showed no effect.

FIG. 5. Cellular localization of proteins of the gp82 family in meta-

cyclic forms of the T. cruzi G strain. Live or permeabilized parasites

were reacted with MAb 3F6 or polyclonal antibodies to J18 or C03 and

processed for analysis by ﬂow cytometry or for visualization by ﬂuo-

rescence microscopy. Note the similar expression patterns of MAb

3F6-reactive molecules in live and permeabilized parasites, whereas

the molecules recognized by anti-J18 antibodies were in part located

intracellularly, and those reacting with anti-C03 antibodies were asso-

ciated with the ﬂagellum and did not react with live parasites.

FIG. 6. Effect of different proteins and antibodies on host cell in-

vasion by T. cruzi metacyclic trypomastigotes (CL strain). (A) HeLa

cells were incubated with parasites in the absence or presence of the

indicated protein at 40 ␮g/ml. (B) Parasites were incubated with the in-

dicated antibodies for 30 min. After antibodies were washed out, the

parasites were incubated with HeLa cells. In both panels, the incuba-

tion time was 1 h, the cells were stained with Giemsa, and the numbers

of intracellular parasites were counted in at least 500 cells. Values in

panel A are the means Ϯ standard deviation of three independent

assays performed in duplicate. The difference between the control and

the cells incubated with J18 protein was signiﬁcant (P Ͻ 0.0005). In

panel B the values are the means Ϯ standard deviation of triplicates of

one representative experiment. A signiﬁcant difference between the

control and the parasites incubated with MAb 3F6 (P Ͻ 0.0005) or

with anti-J18 antibodies (P Ͻ 0.005) was found.

3268 ATAYDE ET AL. I

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DISCUSSION

Our study provides novel information on the expression and

cellular localization of gp82 family members in T. cruzi meta-

cyclic trypomastigotes. This was achieved through the isolation

of a cDNA clone encoding a member of the gp82 family dis-

tinct from the previously characterized cDNA clone selected

for its reactivity with MAb 3F6 (1). From these cDNA clones

encoding proteins with 59.1% identity, we generated recombi-

nant proteins C03 and J18 as well as the corresponding anti-

bodies (Fig. 1 and 3), which served to identify and localize the

gp82 molecules in the parasites.

As a common characteristic, the members of the gp82 family

recognized by MAb 3F6, anti-J18, or anti-C03 antibodies had

isoelectric points at the acidic pH range, with the CL and G

strain molecules focusing at pH 4.6 to 5.4 and at 4.9 to 5.7,

respectively. Otherwise, the gp82 molecules reacting with any

of these antibodies showed considerable differences, particu-

larly in their cellular localization. We conﬁrmed that the MAb

3F6-reactive molecules are present predominantly on the sur-

face of metacyclic trypomastigotes of both the CL and G

strains (Fig. 4 and 5). In T. cruzi strains that enter host cells in

a gp82-mediated manner, the MAb 3F6-reactive molecules

contribute to the invasion process (20), primarily by binding to

the host cell surface molecules and triggering the activation of

signal transduction pathways in both the parasite and target

cells (32). On the other hand, the gp82 molecules recognized

by anti-J18 antibodies appear to be expressed at low levels on

the parasite surface and therefore may have a more marginal

role, if any, in parasite internalization. In CL strain metacyclic

trypomastigotes, which engage surface gp82 molecules to in-

vade target cells (20, 22), the intensity of the reaction of live

parasites with anti-J18 antibodies was much reduced compared

to that with MAb 3F6 (Fig. 4). If MAb 3F6-reactive compo-

nents are included in the gp82 repertoire identiﬁed by anti-J18

antibodies, then the gp82 population present on the surface

that is speciﬁcally recognized by these polyclonal antibodies

may be even smaller. Although to a lesser degree, the intensity

of reaction of live metacyclic forms of G strain with anti-J18

antibodies was also found to be weaker than with MAb 3F6,

but in permeabilized parasites the intensity of reaction was

comparable to that with the MAb (Fig. 5). Taking these results

together, we provide experimental evidence for the existence

of several subpopulations among the gp82 metacyclic trypo-

mastigote proteins, including those that are not targeted to the

cell membrane.

What was particularly striking and unpredicted was the ﬁnd-

ing that members of the gp82 family may be localized to the

ﬂagellum in T. cruzi in a strain-dependent manner. That the

proﬁle of reactivity of metacyclic trypomastigotes with anti-

C03 antibodies could be quite different from that with anti-J18

antibodies was expected, as they were elicited by proteins with

considerable sequence diversity (Fig. 1). However, that gp82

family members could have such different cellular localizations

and that this distribution varied depending on the T. cruzi

strain (Fig. 4 and 5) came as a surprise. Anti-C03 antibodies

did not react with live G strain metacyclic forms but reacted

strongly with components associated with the ﬂagellum in per-

meabilized parasites (Fig. 5), indicating that they recognize

intraﬂagellar molecules. In the CL strain, the proﬁle of the

reaction of live as well as permeabilized parasites with anti-C03

antibodies was similar to that with anti-J18 antibodies, with no

preferential association with ﬂagellum being observed (Fig. 4).

Several T. cruzi molecules associated to ﬂagellum have been

previously described, among them a group of surface proteins

of the 160-kDa family, belonging to the TS/gp85 superfamily

and sharing 48% identity with gp85 proteins (25, 30). These

proteins were detected on the cell membrane in the ﬂagellar

pocket of bloodstream trypomastigotes (28, 30). Comparison

of C03 and Fl-160 amino acid sequences shows 29% identity,

suggesting that they could share epitopes. Other proteins as-

sociated with T. cruzi ﬂagellum are the ﬂagellar calcium-bind-

ing protein (6), the glycoprotein gp72 (19), and a high-molec-

ular-mass (300 kDa) protein built up mostly by nearly identical

repeats of 68 amino acids arranged in tandem (11). The ﬂagel-

lar calcium-binding protein, which is a ﬂagellum-speciﬁc cal-

cium sensor, is associated with the ﬂagellar membrane via its

N-terminal myristate and palmitate moieties in a calcium-mod-

ulated, conformation-dependent manner (6). In epimastigotes,

gp72 is predominantly membrane associated and located on

the cell surface, being evenly distributed over the cell body and

somewhat concentrated in the proximal region of the ﬂagellum

(14). The morphology of gp72-null mutants was found to be

dramatically different from wild-type parasites, and the normal

attachment of the ﬂagellum to the cell membrane was lost (7).

What role is played by intraﬂagellar gp82 molecules recog-

nized by anti-C03 antibodies remains to be determined.

We also conﬁrmed here the involvement of MAb 3F6-reac-

tive gp82 molecules in target cell invasion. Puriﬁed J18 protein,

as well as MAb 3F6 and anti-J18 antibodies, inhibited meta-

cyclic trypomastigote entry into host cells, whereas C03 protein

and anti-C03 antibodies showed no inhibitory effect, as ex-

pected (Fig. 6). This and the new data provided in this study

contribute to our knowledge of T. cruzi gp82 family members,

which may have functions other than interacting with target

cells. Additionally, the differential cellular localization of gp82

molecules recognized by anti-C03 antibodies in G and CL

strains (Fig. 4 and 5) further reminds us of the remarkable

differences between these two strains that have been described

in previous studies.

ACKNOWLEDGMENTS

This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do

Estado de Sa˜o Paulo and Conselho Nacional de Desenvolvimento

Cientı´ﬁco e Tecnolo´gico, Brazil.

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172 Original article

A recombinant protein based on Trypanosoma cruzi

surface molecule gp82 induces apoptotic cell death in

melanoma cells

Vanessa D. Atayde

, Miriam G. Jasiulioni s

, Mauro Cortez

and

Nobuko Yoshida

Trypanosoma cruzi infection is known to confer resistance

to tumor development in mice, and in-vitro studies have

shown the toxic effects of parasite extracts on cancer

cell cultures. Investigations in which T. cruzi molecules

exhibit antitumor activity have just begun. Here, we used

a tumorigenic cell line Tm5, derived from mouse

melanocytes melan-a, to test the effect of J18, a

recombinant protein based on T. cruzi surface molecule

gp82 fused to glutathione-S-transferase (GST). J18

induced actin cytoskeleton disruption in Tm5 but not in

melan-a cells. Several changes indic ative of apoptosis

were detected in Tm5 melanoma cells but not in

melan-a cells treated with J18, such as the flipping of

phosphatidylserine from the inner to the external side

of the plasma membrane, altered nuclear morphology,

DNA fragmentation, increase in mitochondria

depolarization, and in caspase-3 activity. Retention of

NF-jB in the cytoplasm was another alteration observed

specifically in J18-treated Tm5 cells. No such alterations

were found in Tm5 cells treated with GST. In-vivo

experiments showed that C57BL/6 mice inoculated with

Tm5 cells, treated at the site of tumor cell inoculation with

J18, developed tumors of smaller size than mice treated

with phosphate-buffered saline or GST and survived

longer. Melanoma Res 18:172–183

c

2008 Wolters Kluwer

Health | Lippincott Williams & Wilkins.

Melanoma Research 2008, 18:172–183

Keywords: surfac e molecule gp82, melanoma cells, Trypanosoma cruzi

Department of Microbiology, Immunology and Parasitology and

Department of

Pharmacology, Federal University of Sa

o Paulo, Sa

o Paulo, Brasil

Correspondence to Nobuko Yoshida, Escola Paulista de Medicina,

Universidade Federal de Sa

o Paulo, Rua Botucatu, 8 62-61 Andar, 04023-062,

o Paulo, SP, Brasil

Tel: + 55 115 576 4532; fax: + 55 115 571 1095;

e-mail: [email protected]

Received 9 October 2007 Accepted 27 February 2008

Introduction

The incidence of cutaneous malignant melanoma, the

most aggressive form of skin cancer, has increased in

recent decades in most fair-skinned people throughout

the world [1,2]. Melanoma arises from the malignant

transformation of melanocytes, the pigment-producing

cells that reside in the basal layer of the epidermis,

through a complex process involving genetic as well as

environmental factors. Failure to undergo apoptosis is

among the essential alterations that dictate malignant

growth, which include insensitivity to growth-inhibitory

signals, limitless replicative potential, sustained angio-

genesis, tissue invasion, and metastasis [3]. Strategies

to counterbalance this failure may therefore contribute to

anticancer therapies.

Apoptosis, a physiological process of cell death, is

triggered by signals from either the intracellular or

extracellular milieu and is orchestrated by a suicide

machinery conserved through evolution, in which a

proteolytic system involving a family of proteases known

as caspases is a core component [4]. DNA fragmentation,

exposure of phosphatidylserine in the outer leaflet of the

plasma membrane, and loss of mitochondrial membrane

permeability are among the alterations that accompany

apoptosis. From a number of studies in recent years, the

actin cytoskeleton has emerged as a key regulator of

apoptosis [5]. By preventing actin filament elongation

with cytochalasin D, apoptosis was induced in adherent

epithelial cells [6]. Treatment of cells with cytochalasin

D enhanced apoptosis in human leukemia CMK-7

cells by greatly accelerating caspase-3 activation [7].

Jasplakinolide, a drug that stabilizes and aggregates actin

filaments, was shown to increase apoptosis induced by

cytokine deprivation in CTLL-20 cells, the murine

interleukin 2-dependent T cell line, apparently by acting

upstream of the induction of capsase-3-like activity [8].

As regards the cell death induced by jasplakinolide,

through a caspase-3-like protease pathway, an important

finding was that Jurkat cells, derived from human acute

lymphoblastic T cell leukemia, were more susceptible

than normal nontransformed cells [9].

Resistance to tumors in infections by microbial patho-

gens, including the protozoan parasites Toxoplasma gondii

and Trypanosoma cruzi, has been reported [10,11]. In-vitro

experiments have shown the direct inhibitory effect of

T. cruzi lysates on cultured human breast cancer cells [12]

and sarcoma-180 cells [13]. These antitumor effects

may be because of the proapoptotic activity of T. cruzi

0960-8931

c

2008 Wolters Kluwer Health | Lippincott Williams & Wilkins

components. A T. cruzi-released protein Tc52, when in

fusion with glutathione-S-transferase (GST), exhibited

apoptosis-inducing effect on a human T-cell leukemic cell

line [14]. Other T. cruzi components with proapoptotic

activity include a ceramide-containing glycolipid [15]

and trans-sialidase [16,17]. The mechanisms by which

these parasite molecules induce apoptosis have not been

clarified.

Using a line of nontumorigenic mouse melanocytes,

melan-a [18], and the tumorigenic cell line Tm5, derived

from melan-a [19], we aimed in this study to investigate

the apoptosis-inducing potential of a recombinant pro-

tein, based on T. cruzi surface molecule gp82, fused to

GST. This T. cruzi protein, designated J18, which binds

to mammalian cells in a receptor-mediated manner, has

been shown to trigger Ca

-dependent F-actin depo-

lymerization in epithelial HeLa cells [20]. We found out

that J18 induces actin cytoskeleton disruption in Tm5

but not in melan-a cells, leading to the apoptotic cell

death involving caspase-3 activity.

Materials and methods

Mice

Six-week-old female C57BL/6 mice from the animal

facility CEDEME, at Universidade Federal de Sa

o Paulo,

were used for tumor-development experiments. All

procedures and experiments conformed to the regula-

tions of the institutional Ethical Committee for animal

experimentation.

Cell culture, proliferation and adhesion assays

The nontumorigenic murine melanocyte cell line melan-a

[18] was cultured in RPMI 1640 medium (Invitrogen,

Carlsbad, California, USA), pH 6.9, supplemented with

5% fetal calf serum and 200 nmol/l of 4a-phorbol

myristate 13-acetate (Sigma, St Louis, Missouri, USA)

at 371C in a humidified 5% of CO

atmosphere. The

anoikis-resistant tumor cell line Tm5, derived from

melan-a cells and selected upon apoptosis of melan-a

cells by adhesion blockade, as reported in an earlier study

[19], was cultured in the same conditions, without

4a-phorbol myristate 13-acetate. For cell proliferation

studies, 2.5 Â 10

cells were grown at 371C in 96-well

microtiter plates for different periods of time, depending

on the experiment. The number of viable cells was

estimated using a standard methyl thiazol tetrazolium

(MTT) assay (Sigma) and by reading the optical density

at 570 nm, after solubilization of formazan product with

isopropanol [21]. Adhesion assays were performed by

adding melan-a or Tm5 cells (1 Â10

per well) to 96-well

plates. After 20 min of incubation at 371C, washings with

phosphate-buffered saline (PBS) to remove nonadherent

cells, the viable adherent cells were quantified by MTT

assay.

Purification of J18 and gluthatione-S-transferase

The recombinant protein J18, containing the full-length

T. cruzi gp82 sequence (GenBank access number L14824)

in frame with GST, was produced in Escherichia coli (DH5-

a) by transforming the bacteria with a pGEX-3 construct

comprising the gp82 gene [22]. Purification of J18 protein

was performed as previously described [20]. To ascertain

that the correct protein was obtained, the purified sample

was analyzed by silver staining of sodium dodecyl sulfate–

polyacrylamide gel electrophoresis gel, as well as by

immunoblotting using monoclonal antibody 3F6 directed

to gp82. The same procedure to purify J18 was used to

obtain GST.

Preparation of antibodies to J18

To obtain antibodies to the recombinant protein J18,

BALB/c mice were injected intraperitoneally with the

protein (5 mg/mouse) adsorbed in aluminum hydroxide

as adjuvant. Two weeks after the first dose, the animals

received three additional weekly doses of the protein

(5 mg/mouse) in the same adjuvant. The mice were bled

by heart puncture 10 days after the last dose, the sera

were collected and stored at – 201C until used.

Binding of J18 to cells and actin filaments visualization

For binding assay, melan-a or Tm5 cells (3 Â 10

cells per

well) were grown overnight in 96-well plates. After

fixation with 3.5% formaldehyde in PBS, blocking with

PBS containing 10% fetal calf serum, and incubation with

J18 or GST, for 1 h at 371C, the cells were sequentially

incubated with polyclonal antiserum directed to J18 or to

GST, and with peroxidase-conjugated anti-mouse IgG.

The bound enzyme was revealed using o-phenylene-

diamine as detailed elsewhere [23]. For actin filament

visualization, melan-a or Tm5 cells (5 Â 10

), grown in

13-mm diameter round glass coverslips placed in 24-well

plates, were incubated in absence or in the presence of

J18 or GST for 8 h. After fixation with 3.5% formaldehyde

in PBS, the cells were incubated for 1 h at room

temperature with phalloidin-rodhamin (Sigma) in PBS

containing 0.15% gelatin and 0.1% saponin, before

microscopic visualization. Images of stress fibers were

taken with a confocal system (Zeiss Axiovert 100 mol/l),

63 Â1.3 oil objective, using the software LSM 510 Expert

Mode SP2 (Carl Zeiss MicroImaging Inc., Thornwood,

New York, USA).

Determination of caspase-3 activity

Melan-a or Tm5 cells (1 Â 10

cells/well), grown over-

night at 371C in six-well plates, were treated or not with

0.8 mmol/l J18 or GST, at 371C for 36 h, and then

trypsinized for detachment from the substrate. The

activity of caspase-3 in whole cell lysates (2 Â 10

cells/

sample) was determined using colorimetric assay kits

(Calbiochem, UK), according to the manufacturer’s

instructions. Briefly, cells were lysed and centrifuged,

at 17 400g for 10 min at 41C, to remove the cell debris.

Trypanosoma cruzi surface molecule gp82 Atayde et al. 173

The supernatants were incubated for 6 h with 0.2 mmol/l

substrate for caspase-3 (p-nitroanilide-conjugated DEVD

peptide). Human recombinant caspase-3 served as

positive control and cell extracts treated with 0.1 mmol/l

of inhibitor for caspase-3 (DEVD-CHO) served as

negative control. The absorbance was measured at

405 nm using a 96-well colorimetric plate reader (Lab-

systems Multiskan MS, Helsinki, Finland).

Annexin-V assay for detection of phosphatidylserine,

tetramethyl-rhodamine ethyl ester assay for detection

of mitochondrial depolarization and cell-cycle analysis

Melan-a or Tm5 cells (1 Â 10

cells/well), grown in six-well

plates, were treated or not with 0.8 mmol/l of J18 or GST.

After 48 h or 72 h at 371C, depending on the experiment,

cells were trypsinized, collected, washed with PBS, and the

concentration adjusted to 1 Â 10

cells/ml. For phosphati-

dylserine (PS) detection, to 100 mlofcellsamples1mgof

annexin-V-FITC (Santa Cruz Biotechnology, California,

USA) and 10 mlof50mg/ml propidium iodide (PI; Sigma)

were added to 100 ml of cell samples. After incubation for

15 min at room temperature in the dark, flow cytometric

analysis was performed in a FACSCalibur flow cytometer

(Becton Dickinson Biosciences, Palo Alto, California, USA).

Data acquisition and analysis were performed by CellQuest

Pro software (BD Biosciences). The fluorescence intensity

of cells stained with annexin-V-FITC was detected in FL1

channel (525 nm), and PI was detected in the FL3 channel

(620 nm). To detect mitochondrial depolarization, the

mitochondria specific dye tetramethyl-rhodamine ethyl

ester (TMRE) (Molecular Probes-Invitrogen) was added

to 100 mlofcellsamples,kindlyprovidedbyDrSoraya

Smaili, at 100 nmol/l final concentration. After 20-min

incubation at 371C, flow cytometric analysis and data

acquisition were performed as above, by measuring the

fluorescenceofcellsstainedwithTMREinFL2channel

(575 nm). For cell-cycle analysis, cells were fixed with

ethanol (Merck, Darmstadt, Germany), washed with PBS

and treated with 100 mg/ml RNAse A (Sigma) in 100 mlPBS

for 5 min at room temperature. The cells were then

incubated with 50 mg/ml of PI for 30 min at 41C. Flow

cytometric analysis and data acquisition were performed as

in other two assays, by measuring the DNA content

of cells stained with PI in the FL2 channel (575 nm). The

number of apoptotic cells was determined by evaluating

the percentage of hypodiploid nuclei in the DNA less than

2N peak (sub-G1 population). In all three assays 10 000

events were collected from each sample.

Genomic DNA electrophoresis

Melan-a or Tm5 cells (1 Â 10

cells/well), grown in six-

well plates were treated or not with 0.8 mmol/l of J18 or

GST, for 72 h at 371C, before trypsinization. After washes

in PBS, cells were resuspended in TELT buffer (50 nmol/l

Tris, pH 8.0; 62.5 nmol/l EDTA, pH 9.0; 2.5 mol/l LiCl;

4% Triton X-100) and treated with 50 mg of RNAse A

(Sigma) for 1 h at 371C. After addition of 150 mlof

phenol : chloroform : isoamyl alcohol (USB Co, Cleveland,

Ohio, USA), the samples were centrifuged at 17 400g for

15 min. The aqueous phase, containing genomic DNA, was

transferred to new tube and 300 ml of absolute ethanol

(Merck) was added to each sample. After the DNA

precipitation for 20 min at – 701C, the samples were

centrifuged at 17 400g for15min,washedin70%ethanol,

dried, and resuspended in TE buffer (10 mmol/l Tris,

1 mmol/l EDTA, pH 8.0). The extracted DNA was applied

in a 0.8% agarose gel, subjected to electrophoresis and the

ethidium bromide bands were visualized under UV light.

Visualization of the apoptotic cell nucleus and cellular

localization of NF-jB

For both assays, melan-a or Tm5 cells (5 Â 10

), grown in

13-mm diameter round glass coverslips in 24-well plates,

were treated or not with 0.8 mmol/l of J18 or GST. After

48 h or 72 h, depending on the experiment, the cells

were fixed with 3.5% formaldehyde in PBS for 1 h at

room temperature with 4

,6-diamidino-2-phenylindole

(200 mmol/l) in PBS containing 0.15% gelatin with 0.1%

saponin. Images of nuclei were taken at fluorescence

microscope using ACT-1 software. To detect the localiza-

tion of NF-kB, the cells were incubated with anti-NF-kB

monoclonal antibody (1 : 10) (Santa Cruz Biotechnology)

in the PBS containing 0.15% gelatin with 0.1% saponin,

followed by incubation with FITC-conjugated anti-mouse

IgG (Sigma) for 1 h at room temperature, before micro-

scopic visualization and capturing of images as above.

In-vivo tumor growth assay

Tm5 cells were harvested from trypsin-treated subcon-

fluent monolayers, counted, resuspended in PBS and 2 Â

cells were subcutaneously injected into the dorsal

area of 6–8-week-old female C57BL/6 mice, which were

then divided into four groups (n = 8 per group). Starting

on the day of tumor cell inoculation, each group received

either PBS, GST or J18, for 10 consecutive days, at the

site of Tm5 cell injection, except one group that received

J18 on the opposite side. The dose of J18 or GST was

1 mg/kg/day. Tumor size was monitored everyday and

determined as follows: [maximum diameter Â (minimum

diameter)

]/2. Mice with a tumor mass larger than

10 mm

were considered positive for the presence of tumor.

Trypanosoma cruzi strains

T. cruzi strains G and Y* were used to infect mice. Y*

strains are a gp82-deficient variant of Y strain. The

parasites were maintained cyclically in mice and in liver

infusion tryptose medium containing 5% fetal bovine

serum. Metacyclic forms from cultures at the stationary

growth phase were purified by passage through DEAE-

cellulose column, before inoculation into mice.

Statistical analysis

For in-vivo experiments, statistical analysis was per-

formed using the two-way analysis of variance test

17 4 Melanoma Research 2008, Vol 18 No 3

followed by Bonferroni post-tests in the GraphPad Prizm

5.0 Software. To analyze the results of in-vitro experi-

ments, the program GraphPad InStat tm was used to

determine significance by Student’s t-test.

Results

The recombinant protein J18 inhibits the growth

of Tm5 melanoma cells

Melan-a melanocytes and melanoma lineage Tm5 were

treated for 48 h with varying concentrations of J18, which

contains the T. cruzi gp82 sequence fused to GST, and

the cell viability was determined by MTT assay. J18

displayed a significant inhibitory effect on the prolifera-

tion of Tm5 cells, but not of nontumorigenic melan-a

cells, at all concentrations tested (Fig. 1a). At 0.8 mmol/l,

the inhibition by J18 was higher than 60%, and this

concentration was used in all subsequent experiments.

GST was devoid of inhibitory effect. As the growth curves

show (Fig. 1b), the inhibition of Tm5 cell proliferation by

J18 became evident from 48 h postincubation, and by 72 h

the cells were dead.

When visualized using the contrast phase microscope,

J18-treated Tm5 cells, but not melan-a cells, displayed an

altered morphology, with cytoplasm shrinkage and

detachment from neighboring cells as well as from the

substrate (Fig. 1c). The effect of J18 on human tumor

cell lines was also tested. Proliferation of human

melanoma Mel85 cells, as well as of mammary adeno-

carcinoma MCF7 cells, was inhibited more than 60% after

72 h of incubation with J18. Growth inhibition of normal

mammary epithelial MCF10 cells by J18 was negligible.

Treatment of Tm5 melanoma cells with J18 leads to

disassembly of actin fibers and reduced substrate

adhesion

In cell binding assays, J18 bound to melan-a or Tm5 cells

in a dose-dependent manner (Fig. 2a), indicating that the

lack of growth inhibitory effect on melan-a cells (Fig. 1a

and b) is not the result of the differential binding. It has

previously been shown that J18 induces disruption

of actin cytoskeleton in HeLa cells [20]. To determine

whether such an effect was observed in Tm5 cells, the

architecture of actin cytoskeleton was visualized at

different time points after incubation with 0.8 mmol/l

J18, by staining with phalloidin-rhodamine. Within

30 min, F-actin rearrangement was detectable in Tm5

cells, which displayed diminished number of stress fibers,

the alterations becoming more pronounced as the

incubation time increased, whereas GST displayed no

such effect (Fig. 2b). Melan-a cells remained unaltered

by incubation with J18 (Fig. 2b). Treatment of cells with

J18 for 15 min before seeding into microtiter plates,

followed by 20 min of incubation to allow cell attachment,

resulted in diminished adhesion of Tm5 but not of

melan-a cells (Fig. 2c).

J18 induces apoptotic cell death of Tm5 melanoma cells

The F-actin-disrupting effect of J18 observed in HeLa

cells, the epithelial derived from human carcinoma, albeit

to a lesser degree, is similar to that of cytochalasin D [20].

As apoptosis induced by cytochalasin D has been reported

to occur in adherent epithelial cells [6] and also in human

megakaryoblastic leukemia CMK cells [7], we examined

whether the death of Tm5 cells treated with J18 occurred

by apoptotic process. We performed the assay using

FITC-labeled Annexin-V, a Ca

-dependent phospho-

lipid-binding protein with high affinity for PS, which is

located in the inner side of the plasma membrane and

translocates to the external surface in the early stages of

apoptosis [24]. For cell staining, PI with red fluorescence

Fig. 1

Viable cells (OD

570

)

0.80.40.20.10

J18Control(c)

J18 (μmol/l) Time (h)

Tm5 Melan-a Viable cells (%)

100

∗

Tm5

967248240

J18

GST

Control

(b)(a)

Melan-a

150

Growth inhibitory effect of J18 on Tm5 melanoma cells. (a) Melan-a or

Tm5 cells were incubated with different concentrations of J18 for 48 h

and viable cells were quantified by methyl thiazol tetrazolium assay. Tm5

growth inhibition was significant (*) at all J18 concentrations (P < 0.005

at 0.1 mmol/l and P < 0.0001 at 0.8 m mol/l). The reference value of

100% was ascribed to the untreated control. Values are mean ± SD of

three experiments performed in triplicates. (b) Cells were incubated or

not with J18 or glutathione-S-transferase (GST), at 0.8 mmol/l, for

different periods of time. Inhibition of Tm5 cell proliferation was highly

significant at 72 h and 96 h (P < 0.0001). Open and closed symbols

correspond respectively to melan-a and Tm5 growth curves.

Representative results of five experiments, expressed as the mean ± SD

of triplicates, are shown. (c) Phase contrast images showing

morphological changes in Tm5 cells treated or not with 0.8 mmol/l J18

for 72 h. Note the cytoplasm shrinkage and the detachment from the

neighboring cells. No alterations were seen in J18-treated melan-a

cells. Images are representative of four independent assays.

Trypanosoma cruzi surface molecule gp82 Atayde et al. 175

was used simultaneously, to discriminate apoptotic cells

from necrotic cells. Analysis by flow cytometry showed an

increased number of FITC

and PI-negative Tm5 cells

after treatment with J18 for 48 h (Fig. 3), indicating that

PS flipped without membrane damage. To a lesser

degree, there was also an increase in double positive

J18-treated Tm5, which probably represent a minor cell

population at more advanced apoptotic process, in which

the integrity of the membrane was lost. PS exposure was

not observed in melan-a cells treated with J18, or in cells

incubated with GST. Other changes associated with

apoptosis were also found specifically in J18-treated Tm5

cells. An increase in sub-G1 fraction (Fig. 4a), which

is associated with DNA fragmentation (Fig. 4b), was

detectable after 72 h of incubation of Tm5 cells with

J18. No such alteration was detected in melan-a cells

treated with J18 (Fig. 4a and b). The nuclei of

J18-treated Tm5 cells, examined by staining with DAPI,

exhibited altered morphology, aggregated chromatin and

blebs (Fig. 4c).

Fig. 2

Melan-a

J18

1.0

(c)(a)

0.5

0.0

Control GST J18

GST

Tm5

Melan-a

Tm5

∗

1.2

0.6

Cell binding (OD

492

)Melan-aTm5

Substrate adhesion (OD

570

)

0.0

0 0.1 0.2

Protein (μmol/l)

Control(b) GST J18

0.4 0.8

J18-induced disassembly of actin fibers in Tm5 melanoma cells and reduced adhesion to substrate. (a) Melan-a or Tm5 cells were fixed and

incubated with varying concentrations of J18 or glutathione-S-transferase (GST). The binding was revealed by sequential incubation with polyclonal

antiserum directed to J18 or to GST and with peroxidase-conjugated anti-mouse IgG. Note the similar binding profile of J18 to both cells. The

representative results of three independent assays performed in triplicate are shown. Values are the mean ± SD of triplicates. (b) Confocal images

showing the phalloidin-rodhamin-stained actin filaments of melan-a or Tm5 cells incubated or not for 8 h with 0.8 mmol/l of J18 or GST. Note the

stress fibers decrease in Tm5 cells treated with J18, as compared with controls or to melan-a cells. Scale bar: 10 mm. Images are representative of

four independent experiments. (c) Substrate adhesiveness of melan-a or Tm5 cells treated or not for 15 min with 0.8 mmol/l of J18 or GST. Pretreated

cells were added to microtiter plates and incubated for 20 min at 371C for attachment. After removing nonadherent cells, adherent cells were

quantified by methyl thiazol tetrazolium assay. The differential adhesiveness of Tm5 cells treated with J18 was significant (P < 0.02). Data are the

mean ± SD of triplicates and representative of three independent experiments.

176 Melanoma Research 2008, Vol 18 No 3

Caspase-3 activity is increased in J18-treated Tm5

melanoma cells

We checked the involvement of caspases in the apoptotic

cell death of Tm5 cells, in particular of the effector

caspase-3, which has its activity increased upon treat-

ment of transformed cells with cytochalasin D [7].

Melan-a or Tm5 cells were incubated for 36 h with J18

or GST, and the activity of caspase-3 was measured.

In J18-treated Tm5 cells, but not in melan-a cells, there

was an increase in caspase-3 activity, which was

inhibitable by a specific inhibitor (Fig. 5). Provided that

mitochondria regulate the final execution of the apoptotic

program by releasing apoptogenic factors [25–27], and

cytochrome c-related caspase-3 activation have been

reported [28], we investigated the involvement of these

organelles at late stages of the Tm5 melanoma apoptotic

Fig. 3

Annexin-V

Tm5

ControlGSTJ18

Propidium iodide

8.00% 7.47%

0.49%

7. 4 8 %

54.69%

19.39%2.23%8.21%2.98%

0.51%

1.41%

9.62%8.31%2.47%5.60%

0.36%

1.44%

8.77%

Melan-a

Phosphatidylserine exposure on Tm5 melanoma cells treated with J18. Melan-a or Tm5 cells were treated or not with 0.8 mmol/l of J18 or glutathione-

S-transferase (GST) for 48 h. Cells were collected and incubated with annexin-V-FITC and propidium iodide (PI) for 15 min at room temperature in

the dark. Flow cytometric analysis was performed on a FACSCalibur flow cytometer. The fluorescence of cells stained with annexin-V-FITC was

detected in FL1 channel (525 nm), whereas PI was detected in the FL3 channel (620 nm). Ten thousand events were collected from each sample.

Graphics shown are representative of three independent experiments.

Trypanosoma cruzi surface molecule gp82 Atayde et al. 177

pathway. By using the fluorescent probe TMRE, which

accumulates rapidly and reversibly in mitochondria [29],

we examined the loss of mitochondria transmembrane

potential, which is one of the events in apoptosis [30].

In Tm5 cells treated with J18 for 72 h, but not in

J18-treated melan-a cells or GST-treated cells, mito-

chondrial depolarization was detected to some extent

(Fig. 6).

Translocation of NF-jB to the nucleus is reduced

in Tm5 melanoma cells treated with J18

Constitutive activation of the nuclear transcription factor

NF-kB, which is normally sequestered in the cytosol and

translocates to the nucleus upon degradation of its

inhibitor IkB, is an emerging hallmark of various types

of tumor, including melanoma [31]. Nuclear localization

and NF-kB-binding to DNA is enhanced in melanoma

Fig. 4

10008006004002000

100

10008006004002000

100

10008006004002000

100

Tm5 J18

Tm5 GST

Tm5 control

M-a J18

M-a GST

M-a control

Propidium iodide

1000

8006004002000

0.1

0.4

2.0

4.3

6.5

23.1

100

10008006004002000

Cell counts

100

10008006004002000

3.6%

0.8%

62.1%

18.5%

2.6%

J18 GST Control

Tm5

(c)(b)

(a) Melan-a

0.7%

100

Increase in sub-G1 population in Tm5 melanoma cells treated with J18. (a) Cell-cycle analysis of melan-a or Tm5 cells treated or not with 0.8 mmol/l of

J18 or glutathione-S-transferase (GST) for 72 h. Fixed cells were stained with propidium iodide and analyzed by flow cytometry. The DNA content of

cells was detected in the FL2 channel (575 nm). The number of DNA-fragmented cells was determined by evaluating the percentage of less than 2N

DNA peak (sub-G1 population). Ten thousand events were collected from each sample. The representative of three independent experiments are

shown. (b) Genomic DNA extracted from melan-a or Tm5 cells treated or not with 0.8 mmol/l of J18 or GST for 72 h was applied in a 1.0% agarose

gel and the ethidium bromide bands were seen under UV light. The same profile was seen in two other experiments. (c) Visualization of apoptotic

nuclei of Tm5 cells treated with 0.8 mmol/l J18 for 72 h. Fixed cells were stained with 4

,6-diamidino-2-phenylindole and images were obtained at

fluorescence microscope utilizing the ACT-1 software. Representative images: (1: kidney shaped; 2: chromatin aggregation; 3: nuclear blebbing; 4:

nuclear blebs) of three independent experiments are shown.

178 Melanoma Research 2008, Vol 18 No 3

cells, as compared with normal melanocytes [32]. Here,

we examined whether treatment of Tm5 cells with

J18 interfered with nuclear localization of NF-kB. Melan-

a and Tm5 cells were incubated with J18 for 48 h and

then processed for detection of NF-kB using monoclonal

antibody directed to NF-kB subunit p50. The images

visualized using the fluorescence microscope showed a

predominant cytoplasmic localization of NF-kB in J18-

treated Tm5 cells at 48 h postincubation, whereas much

fewer J18-treated melan-a cells were negative for nuclear

NF-kB (Fig. 7).

J18 reduces the tumor growth in mice inoculated with

Tm5 melanoma cells

Experiments were also performed to test the antitumor

activity of J18 in vivo. C57BL/6 mice were injected

subcutaneously with Tm5 cells in the dorsal area and

then separated in four groups. For 10 consecutive days,

starting on the day of Tm5 cell inoculation, one group of

mice was treated with PBS, the second group with GST,

the third group with J18, all of them at the site of

Fig. 6

100

12.1%

Melan-a Tm5

21.3%

23.8%

100

Cell counts

ControlGSTJ18

15.1%

100

17.2%

TMRE

100

38.8%

Reduction of mitochondrial polarity in Tm5 melanoma cells treated with J18. Melan-a or Tm5 cells, treated or not with 0.8 mmol/l of J18 or glutathione-

S-transferase (GST) for 72 h, were collected and incubated with tetramethyl-rhodamine ethyl ester (TMRE) for 20 min at 371C. Flow cytometric

analysis was performed in a FACSCalibur flow cytometer. The fluorescence of cells stained with TMRE was detected in FL2 channel (575 nm).

Ten thousand events were collected from each sample. Shown are the representative results of three independent experiments.

Fig. 5

0.4

0.2

Caspase–3 activity (OD

405

)

0.0

Melan-a Tm5

Control

GST

J18

J18+C3-I

Caspase-3 activation in Tm5 melanoma cells treated with J18. The

activity of caspase-3 in whole cell lysates was determined using a

colorimetric assay. Cells incubated or not for 36 h with 0.8 mmol/l J18

or glutathione-S-transferase (GST) were lysed. Supernatants were then

incubated with caspase-3 substrate (DEVD-p-nitroanilide) or with

caspase-3 substrate in the presence of caspase-3 inhibitor (C3-I/

DEVD-CHO), for 6 h at 371C. The absorbance of each sample was

measured at 405 nm. Data are the means of triplicates from one out

of three independent experiments.

Trypanosoma cruzi surface molecule gp82 Atayde et al. 179

Fig. 7

Control

NF-κBNF-κBDAPI DAPI

Melan-aTm5

J18

NF-kB localization in Tm5 melanoma cells treated with J18. Melan-a or Tm5 cells were incubated or not for 48 h with 0.8 mmol/l of J18 or glutathione-

S-transferase (GST), fixed and incubated sequentially with anti-NF-kB monoclonal antibody, FITC-conjugated anti-mouse IgG and 4

,6-diamidino-2-

phenylindole (DAPI). Note the predominant cytoplasmic localization of NF- kB in Tm5 cells treated with J18. No alterations were seen in cells treated

with GST. Images, obtained with the ACT-1 software, are representative of three independent experiments. White and black arrows point to nuclei

negative and weakly positive for NF-kB, respectively.

Fig. 8

6000

Control

GST

J18 control

J18

Tumor volume (mm

)

Tumor volume (mm

)

5000

4000

3000

2000

1000

Time (days)

5000

(b)

(a)

100

0102030

Time (days)

40 50

PBS

J18 (8− 23)

J18 (1− 10)

4000

3000

2000

1000

9111315

Time (days)

17 19 21 23

12 14 16 18 20

100

Percent survival

01020

Time (days)

30 40

Reduction of in-vivo tumor growth in mice treated with J18. (a) Tm5 cells (2 Â 10

) were injected subcutaneously into the dorsal area of C57BL/6

mice ( n = 8 per group). The mice were divided into four groups: phosphate-buffered saline (PBS) treated, glutathione-S-transferase (GST) treated,

J18 treated outside the tumor area (J18 control), and J18 treated in tumor area (J18). Treatment with J18 or GST (1 mg/kg/day) was performed for 10

consecutive days beginning on the day of melanoma cell inoculation. Development of tumor is shown on the left and mortality on the right. Data are

the mean ± SD of tumor volumes of eight animals from each group, and are representative of three independent experiments. The tumor growth

inhibition in J18 group from day 17 to 20 was significant (P < 0.001). (b) C57BL/6 mice were injected subcutaneously with Tm5 cells (2 Â 10

) and

separated in three groups (n = 5 per group): PBS treated, J18 treated in tumor area starting on day zero of melanoma cell inoculation, and J18

treated from day 8 to 23. Data are the mean ± SD of tumor volumes of five animals from each group. Inhibition of tumor growth in J18 group was

significant from day 21 to 23 (P < 0.001).

180 Melanoma Research 2008, Vol 18 No 3

melanoma cell injection, whereas the fourth group

received J18 on the opposite side of Tm5 cell inoculation.

The daily dose of either of J18 or GST was 1 mg/kg/day.

Of the four groups, only the mice inoculated with J18

proximal to the site of melanoma cell injection developed

tumors of smaller size than the controls and survived

longer (Fig. 8a). We confirmed that J18, by itself, is not

harmful to the animals. When injected daily with J18,

following the same procedure for the experiments

described above, the mice remained healthy during a

period of 60 days, without signs of harmful effects, as if

they had not received any treatment. To test the effect of

J18 in vivo on more established melanoma, a group of mice

was inoculated with tumorigenic Tm5 cells and subjected

to treatment with J18 starting on day 8 postinoculation.

This scheme was as effective in delaying tumor develop-

ment as the protocol of treatment with J18 starting on the

day of Tm5 cell inoculation (Fig. 8b).

Tumor development is slower in mice infected with

T. cruzi strain expressing high levels of gp82 than in

mice infected with gp82-deficient strain

In addition to the effect of J18, we also investigated

whether mice inoculated with metacyclic forms of T. cruzi

strains expressing or deficient in gp82, the surface

molecule on which J18 is based, displayed differential

tumor development after injection of Tm5 cells. C57BL/

6 mice were separated in three groups. One of them was

inoculated intraperitoneally with metacyclic forms (10

mouse) of T. cruzi strain G, which express high gp82 levels

on the surface [33], whereas the other group received

metacyclic forms of strain Y*, which are deficient in gp82

(Fig. 9a). Three weeks postinfection, all groups were

injected subcutaneously with Tm5 cells in the dorsal

area. Tumor development was slower in mice infected

with G strain as compared with mice infected with Y*

strain or uninfected controls (Fig. 9b).

Discussion

Our study shows that a recombinant protein, J18, based

on the T. cruzi surface molecule gp82, is capable of

specifically inducing the apoptotic cell death of melano-

ma cells without exerting any effect on normal melano-

cytes. Since the early findings, more than 70 years ago,

that T. cruzi infection conferred resistance to tumor

development in mice, studies using killed parasites or

their extracts either in cancerous animals or in-vitro

cancer cell cultures have been performed, with con-

troversial results [12,13,34]. Although the possibility of T.

cruzi factors with direct toxic activity toward tumor cells

has been raised, investigations on the antitumor proper-

ties of molecularly defined T. cruzi components are just

beginning. Recently, a T. cruzi protein Tc52 fused to GST

was shown to exhibit an apoptosis-inducing effect on a

human T-cell leukemic cell line [14]. Other T. cruzi

molecules, such as the ceramide-containing glycolipid

and trans-sialidase were reported to have proapoptotic

activity toward cells of the immune system [15–17]. It

remains to be determined whether they possess anti-

tumor activity.

What is relevant about the effect of J18, the protein based

on T. cruzi surface molecule gp82, which also acts in vivo,

is its capacity of inhibiting the growth and ultimately

leading to death the tumorigenic Tm5 cells, but not the

melan-a cells, the normal melanocytes from which they

derived. J18 induced actin cytoskeleton disruption in

Tm5 cells and this is possibly associated with its

apoptosis-promoting activity. Apoptosis of different cell

types, under various conditions, has been shown to occur

after treatment with cytochalasin D or jasplakinolide,

drugs that affect the cellular actin architecture [6–9].

Several evidences indicate that J18 induces apoptosis in

Tm5 cells. Flipping of phosphatidylserine from the inner

to the external side of the plasma membrane was

detected in J18-treated Tm5 melanoma cells but not in

melan-a melanocytes. A number of other events asso-

ciated with the apoptotic process, such as alteration in

nuclear morphology, DNA fragmentation, and decrease in

mitochondria transmembrane potential, were also found

to occur specifically in J18-treated Tm5 cells. The

apoptotic pathway triggered by J18 possibly involves the

executioner caspase-3. In J18-treated Tm5 cells, the

activity of caspase-3 was increased. Another finding that

reinforces the apoptosis-inducing activity of J18 toward

Tm5 cells is that NF-kB is mostly retained in the

Fig. 9

T. cruzi

gp82

(a) (b)

gp30

∗

2000

Control

G strain

∗

strain

1500

1000

Tumor volume (mm

)

500

91113

Time (days)

15 17 19

Tumor development in mice infected with gp82-expressing or gp82-

deficient T. cruzi strains. (a) Metacyclic trypomastigotes of G and Y*

strains were processed for western blot analysis using monoclonal

antibody 3F6 directed to the surface molecule gp82. Note the

expression of high levels of gp82 in G strain and the detection of gp30,

instead of gp82, in Y* strain. (b) Groups of C57BL/6 mice were

separated in three groups (n = 5 per group) and were inoculated with

metacyclic forms (10

/mouse) of T. cruzi strain G or Y*, or remained

uninfected. Three weeks later, all animals were injected subcutaneously

with Tm5 cells (2 Â 10

). Data are the mean ± SD of tumor volumes of

five animals from each group. The delay in tumor development in mice

infected with T. cruzi G strain was significant from day 15 to 18

(P < 0.001).

Trypanosoma cruzi surface molecule gp82 Atayde et al. 181

cytoplasm rather than translocating into the nucleus. The

activation of this nuclear transcription factor is associated

with the growth of various types of tumor, including

melanoma [31,32]. NF-kB activity was found to be

upregulated in lesions of melanoma when compared with

melanocytes in normal skin [32,35,36]. Taken together,

our data suggest that J18 specifically exerts its activity on

Tm5 melanoma cells not only by inducing the apoptotic

process, but and also by inhibiting cell growth through its

negative effect on NF-kB translocation to the nucleus.

Another interesting finding is that in mice infected with

metacyclic trypomastigotes of T. cruzi strain G, but not

of strain Y*, before injection of Tm5 melanoma cells,

a significant delay of tumor development was observed.

The main difference in the surface profile of the infective

metacyclic forms of strains G and Y* is the expression of

gp82, the surface molecule defined by monoclonal

antibody 3F6 [37] on which J18 protein is based. Instead

of gp82, Y* metacyclic forms express the surface

glycoprotein gp30 that also reacts with monoclonal

antibody 3F6 and, as gp82, is involved in host cell

invasion [33]. Gp82 is a member of a large gp85/sialidase

multigene family [38]. Included in this superfamiIy are

Tc-85 and trans-sialidase (TS). In this regard, it is of

interest that TS, similarly to gp82, was shown to have

proapoptotic activity [16,17]. TS also known as SAPA,

which is linked to the outer leaflet of the parasite plasma

membrane through glycophosphatidylinositol anchor

[39], is secreted and can be found in the blood of

T. cruzi-infected patients at the acute phase [40]. Tc85,

a surface glycoprotein expressed in parasite forms

corresponding to the bloodstream trypomastigotes, con-

tains binding sites for laminin [41,42] and is implicated in

mammalian cell entry [43]. To what extent Tc85 exhibits

the signaling properties similar to those displayed by

metacyclic trypomastigote gp82, and whether it could

inhibit tumor development, has not been investigated.

The scenario we envisage when mice infected with

T. cruzi are inoculated with cancer cells is that molecules

of gp85/sialidase family, which are preferentially ex-

pressed in infective trypomastigote forms that circulate

in the blood, act on tumor cells, interfering with their

development through growth inhibition and apoptotic

mechanisms, such as described for J18. Another possibi-

lity is that the immune response elicited by T. cruzi is also

effective toward tumor cells. A third possibility is that

both the toxic activity of T. cruzi factors and the parasite-

induced immune effectors are acting in concert.

Acknowledgements

This work was supported by Fundac¸a

o de Amparo a

Pesquisa do Estado de Sa

o Paulo (FAPESP) and Conselho

Nacional de Desenvolvimento Cientı

fico e Tecnolo

gico

(CNPq), Brasil. The authors thank Dr Erika Suzuki for

critical reading of the manuscript.

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