Trypanosoma cruzi is the etiological agent of Chagas' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction.
Here, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain.
These data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host.
Chagas' disease, a neglected tropical disease caused by Trypanosoma cruzi, is transmitted to vertebrate hosts by hematophagous insects. Cruzipain is a lysosomal cysteine peptidase, which plays an important role in parasite infectivity, intracellular growth and differentiation, and is abundantly expressed on the surface of epimastigotes. Since these forms face the insect vector environment during the life cycle, it is conceivable that cruzipain may participate in the interaction process with the invertebrate host. Here, we showed that adhesion of T. cruzi to the insect midgut cells was inhibited by the blockage of cruzipain function. Cysteine peptidase inhibitors, in a dose-dependent manner, and anti-cruzipain antibodies were able to reduce the binding of epimastigote forms to the Rhodnius prolixus midgut. Similarly, T. cruzi transfectants that overexpress chagasin, the endogenous cruzipain inhibitor, displayed low levels of adhesion. Accordingly, the supplementation of exogenous cruzipain partially restored the adherence of the transfected line. Additionally, the ability of the chagasin overexpressing transfectants to colonize the insect in vivo was drastically reduced, and the levels of cruzipain expression by wild-type parasites were enhanced after in vivo passage in R. prolixus. Collectively, our results strongly suggest that cruzipain is required for successful colonization of R. prolixus by T. cruzi.