Oral Presentation

Abstract

Trypanosoma cruzi relies on cysteine proteases (CPs) for efficient growth and survival inside the host. The activity of the main parasite CP, cruzipain, has been directly or indirectly associated with intracellular amastigote survival, host cell invasion and inflammation. Cruzipain is encoded by a large gene family, whose members present variable polymorphism and are developmentally regulated. Studies on cruzipain expression showed that it is post-transcriptionally regulated, leading to increased levels of CP activity in the replicative stages. Although cruzipain has been extensively characterized at the biochemical and structural level, little is known about the mechanisms controlling its activity in living parasites. Recently, we have identified an endogenous CP inhibitor in T. cruzi, chagasin, which potently inactivates cruzipain, suggesting that it may regulate CP activity in the parasite. Notably, the expression of chagasin in the life stages of T. cruzi is inversely correlated with that of cruzipain. Here we investigate whether cruzipain and chagasin interact in living parasites and also evaluated the levels of functional enzyme and inhibitor in epimastigotes of six T. cruzi isolates. The cruzipain:chagasin complex was visualized by Western blot under partially denaturing conditions, using anti-chagasin antibodies. The detection of the complex immediately after parasite lysis suggests that they may interact in vivo. These complexes were more abundantly detected in the isolates Dm28c, Sylvio X10/6, Y and G, but are poorly detected in the isolates CL and Brazil. Analysis of steady-state levels of cruzipain, using monoclonal antibodies, showed a high expression of this CP in the isolates Dm28c, Sylvio X10/6, but a low expression in Y, CL, G and Brazil parasites. Likewise, quantification of functional enzyme in parasite lysates upon titration with MuPhe-hPhe-VSO (an inhibitor of cruzipain) revealed the occurrence of lower levels of active cruzipain in CL, Brazil and G parasites. Interestingly, although these three isolates presented similar steady-state levels of cruzipain by Western blot, the G isolate bears 3-5 fold less functional cruzipain than the others. Accordingly, the titration of functional chagasin revealed that a reduced concentration of the inhibitor is found in isolates presenting lower cruzipain expression (CL and Brazil), with the exception of the G isolate which showed a high content of chagasin. These observations indicate that in T. cruzi, low cruzipain expression is accompanied by a reduction in the chagasin levels, resulting in a reduced formation of the cruzipain:chagasin complex in living cells. As a consequence, these parasites present only a 2-fold difference in the amount of functional cruzipain when compared to isolates with abundant cruzipain expression (i.e. Dm28c, X10/6, Y). Parasites that do not compensate decreased cruzipain expression by lowering the amount of chagasin (i.e. G) present higher accumulation of cruzipain:chagasin complexes, resulting in 10-fold less functional cr...