Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells

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.

Antigen characterization of vector-borne and cultured metacyclic trypomastigotes of Trypanosoma cruzi

Caracterizaçäo antigênica de tripomastigotas metacíclicos de Trypanosoma cruzi provenientes do vector e de cultura. Tripomastigotas metacíclicos da cepa CL de Trypanosoma cruzi, originários de triatomíneos e de cultura axênica, foram comparativamente analisados quanto a sua composiçäo antigênica e características imunogênicas. Os vários parâmetros examinados mostraram que as duas formas metacíclicas säo 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 contra infecçäo aguda por T. cruzi, por imunizaçäo com formas metacílicas 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 formas metacíclicas 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 reaçäo mediada por complemento

Resistance of mice inmunized with killed culture trypomastigotes against infection by insect-derived trypomastigotes of Trypanosoma cruzi

Camundongos imunizados com tripomastigotas de cultura da cepa G, mortos pelo calor ou mertiolato, mostraram-se resistentes a infeccao por tripomastigotas da cepa CL de T. cruzi provenientes do inseto vetor. Em 90% dos camundongos imunizados nao foi detectada parasitemia patente ao exame microscopico enquanto todos os animais-controle desenvolveram alta parasitemia.Tripsinizacao seguida de aquecimento, ou fixacao com para-formaldeido, aparentemente reduziram a imunogenicidade de tripomastigotas da cepa G, visto que camundongos imunizados com tripomastigotas tratados por qualquer destes metodos nao foram protegidos contra infeccao por T. cruzi.A analise de proteinas de superficie de tripomastigotas da cepa G, inativados por diferentes metodos, marcados com 131I, sugere que esses componentes de superficie estao envolvidos na inducao da imunidade protetora contra T. cruzi