Peptidases do Trypanosoma cruzi e Rhodnius prolixus: implicações funcionais na interação

• Autor(a) principal: Uehara, Lívia Almeida
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/14368
• topic: Trypanosoma cruzi; Gp63; Peptidases; Rhodnius prolixus; Trypanosoma cruzi; Gp63; Peptidases; Rhodnius prolixus; Trypanosoma cruzi; Peptídeo Hidrolases; Rhodnius prolixus; Chagas, Doença de; CNPQ::CIENCIAS BIOLOGICAS::MICROBIOLOGIA::MICROBIOLOGIA APLICADA::MICROBIOLOGIA MEDICA

The highly abundant surface peptidase of Leishmania spp., gp63, is widely distributed in the Trypanosomatidae family and is particularly involved in host-parasite interaction. The gp63 homologues in monoxenic trypanosomatids appears to be essential for nutrition and to interaction with the insect epithelial cells. In this context, this project aims through a biochemical approach to investigate the proteolytic profile of the Trypanosoma cruzi cellular extract, for the detection of gp63 in Dm28c strain, analyze the role of this metallopeptidase in the interaction of T. cruzi with an insect vector, Rhodnius prolixus and characterize the proteolytic profile of the triatomine peptidases. Ultimately, this set of approaches will assess the relevance of the parasite and insect peptidase in the interaction process. To this end, we have analyzed the effects of the treatment of T. cruzi with metallopeptidase inhibitors or anti-gp63 antibodies in the parasite adhesion to R. prolixus posterior midgut ex vivo. The interaction rate was reduced in the presence of 1,10-phenanthroline 0.5 µM (79%), EDTA 1 µM (74%) and EGTA 1 µM (68%) in comparison to control parasites. 1,10-phenanthroline presented a dose-dependent reduction on the adhesion. In addition, anti-gp63 antibodies (1:500) reduced the adhesion to the insect posterior midgut in 65% in comparison to control parasites. There were no significant differences in gp63 and cruzipain exposure when cells were treated with the inhibitor 1,10-phenanthroline for 1 h or 24 h as compared to non-treated cells. On the other hand, the level of gp63 exposure was drastic enhanced after in vivo passage in R. prolixus. In total cellular extract of T. cruzi, three typical halos of gelatin degradation were detected. A most prominent one of approximately 52 kDa, classified as metallopetidase, since it was inhibited in alkaline pH by 1,10-phenanthroline 20 mM, EDTA and EGTA 5 mM. Two other enzymes were detected: another metallopeptidase, presenting a fainter degradation halo, which migrates around 60 kDa, and a very weak cysteine peptidase that resisted the extraction in the presence of specific inhbitor. These results together demonstrate the presence of an active metallopeptidase in the parasite, possibly involved in the interaction with the vector. In relation to the characterization of active peptidases in R. prolixus, the proteolytic profile consist of three bands migrating in the range of 31-24 kDa in adult males and females midgut. Peptidases belong to the cysteine peptidase class (strongly inhibited by E-64), with activity better detected in acid pH (4.5) after 24 h of incubation. Moreover, enzymatic assays with the substrate Z-Phe-Arg-AMC showed the highest level of activity at pH (5.5). Collectively, our results suggest a possible involvement of metallopetidases, particularly gp63, in the interaction between T. cruzi and R. prolixus midgut.