Penetration of Trypanosoma cruzi into mammalian cells depends on the activation of the parasite's protein tyrosine kinase and on the increase in cytosolic Ca2+ concentration. We used metacyclic trypomastigotes, the T. cruzi developmental forms that initiate infection in mammalian hosts, to investigate the association of these two events and to identify the various components of the parasite signal transduction pathway involved in host cell invasion. We have found that i) both the protein tyrosine kinase activation, as measured by phosphorylation of a 175-kDa protein (p175), and Ca2+ mobilization were induced in the metacyclic forms by the HeLa cell extract but not by the extract of T. cruzi-resistant K562 cells; ii) treatment of parasites with the tyrosine kinase inhibitor genistein blocked both p175 phosphorylation and the increase in cytosolic Ca2+ concentration; iii) the recombinant protein J18, which contains the full-length sequence of gp82, a metacyclic stage surface glycoprotein involved in target cell invasion, interfered with tyrosine kinase and Ca2+ responses, whereas the monoclonal antibody 3F6 directed at gp82 induced parasite p175 phosphorylation and Ca2+ mobilization; iv) treatment of metacyclic forms with phospholipase C inhibitor U73122 blocked Ca2+ signaling and impaired the ability of the parasites to enter HeLa cells, and v) drugs such as heparin, a competitive IP3-receptor blocker, caffeine, which affects Ca2+ release from IP3-sensitive stores, in addition to thapsigargin, which depletes intracellular Ca2+ compartments and lithium ion, reduced the parasite infectivity. Taken together, these data suggest that protein tyrosine kinase, phospholipase C and IP3 are involved in the signaling cascade that is initiated on the parasite cell surface by gp82 and leads to Ca2+ mobilization required for target cell invasion.

Tripomastigotas metacíclicos da cepa CL de Trypanosoma cruzi, originários de triatomineos e de cultura axênica, foram comparativamente analisados quanto a sua composição antigênica e características ímunogênicas. Os vários parâmetros examinados mostraram que as duas formas metacíclicas sáo semelhantes. Assim, soros de camundongos imunizados com qualquer uma das formas metacíclicas precipitaram uma proteína de superfície de 82Kd de tripomastigotas metacíclicos de cultura marcados com 131I. Soros de camundongos protegidos con trn infecção aguda por T. cruzi, por imunização com formas metacíclicas de cultura inativadas, de uma cepa diferente (G), detectaram por imunoblotting uma proteína de 77Kd em ambas as formas metacíclicas da cepa CL. Um anticorpo monoclonal, produzido contra as for mas metaciclicas da cepa G, e específico para tripomastigotas metacíclicos, reagiu com formas metacíclicas da cepa CL tanto do vetor quanto de cultura. As duas formas metacíclicas mostraram suscetibilidade semelhante à lise por vários soros anti-T. cruzi, em uma reação mediada por complemento.

Metacyclic trypomastigotes ol the CL strain of Trypanosoma cruzi obtained from triatomid vectors and from axenic cultures were comparatively analysed as to their antigen make-up and immunogenic characteristics. They were found to be similar by the various parameters examined. Thus, sera of mice immunized with either one of the two metacyclic types precipitated a 82Kd surface protein from 131I-labeled culture metacyclics. Sera of mice protected against acute T. cruzi infection by immunization with killed culture metacyclics of a different strain (G) recognized, by immunoblotting, a 77Kd protein in both types of CL strain metacyclics. A monoclonal antibody raised against G strain metacyclics, and specific for metacyclic stages of this strain, reacted with both CL strain metacyclic types. Both metacyclic forms were similarly Iysed by various anti-T. cruzi sera, in a complement-mediated reaction.

Camundongos imunizados com tripomastigotas de cultura da cepa G, mortos pelo calor ou mertiolato, mostraram-se resistentes à infecção por tripomastigotas da cepa CL de T. cruzi provenientes do inseto vetor. Em 90% dos camundongos imunizados não foi detectado parasitemia patente ao exame microscópico enquanto todos os animais controle desen volveram alta parasitemia. Tripsinizaçao seguida de aquecimento, ou fixação com para formal deido, aparentemente reduziram a imunogenicidade de tripomastigotas da cepa G, visto que camundongos imunizados com tripomastigotas tratados por qualquer destes métodos não foram protegidos contra infecção por T. cruzi. A análise de proteínas de superficie de tripomastigotas da cepa G. inativados por diferentos métodos, marcados com 131,I, sugere que esses componentes de superfície estão envolvidos na indução da imunidade protetora contra T. cruzi. Camundongos imunizados com tripomastigotas de cultura da cepa G. mortos pelo calor ou mertiolato. mostraram se resistentes à in fecção por tripomastigotas da cepa CL de T. cruzi provenientes do inseto vetor. Em 90% dos camundongos imunizados não foi detectada parasitemia patente ao exame microscópico enquanto todos os animais controle desen volveram alta parasitemia. Tripsinizaçao seguida de aquecimento, ou fixação com para formal deido, aparentemente reduziram a imunogenicidade de tripomastigotas da cepa G, visto que camundongos imunizados com tripomastigotas tratados por qualquer destes métodos não foram protegidos contra infecção por T. cruzi. A análise de proteínas de superficie de tripomastigotas da cepa G. inativados por diferentos métodos, marcados com 131,I, sugere que esses componentes de superfície estão envolvidos na indução da imunidade protetora contra T. cruzi.

Mice immunized with heat or merthiolate-killed culture trypomastigotes of the non-virulent G strain were resistant to the challenge by insect-derived trypomastigotes of the CL strain of Trypanosoma cruzi. No parasitemia was detected, by direct microscopic examination of blood samples, in 90% of immunized mice while all control animals developed a high parasitemia. Trypsinization before heat-inactivation, or fixation with paraformaldehyde, apparently reduced the immunogenicity of the G strain trypomastigotes. Mice immunized with trypomastigotes treated by either of these procedures were not protected against infection by virulent T. cruzi. Analysis of the 13I-labeled surface proteins of G strain trypomastigotes inactivated by the various methods suggests that these components are involved in eliciting protective immunity against T. cruzi infection.