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Abstract: The enteric protozoan parasite Entamoeba histolytica uniquely possesses two isotypes of ICPs, a novel class of inhibitors for cysteine proteases. These two EhICPs showed a remarkable difference in the ability to inhibit cysteine protease (CP) 5, a well-established virulence determinant, whereas they equally inhibited CP1 and CP2. Immunofluorescence imaging and cellular fractionation showed that EhICP1 and EhICP2 are localized to distinct compartments. While EhICP1 is localized to the soluble cytosolic fraction, EhICP2 is targeted from lysosomes to phagosomes upon erythrocyte engulfment. Overexpression of either EhICP1 or EhICP2 caused reduction of intracellular CP activity, but not the amount of CP, and decrease in the secretion of all major CPs, suggesting that both EhICPs are involved in the trafficking and/or interference with the major CP activity. These data indicate that the two EhICPs, present in distinct subcellular compartments, negatively regulate CP secretion, and, thus, the virulence of this parasite. [Copyright &y& Elsevier]

Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the cruzipain family, we sought to determine whether chagasin and cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and β-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed cruzipain-chagasin complexes, we obtained direct: evidence that chagasin-cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble cruzipain activity and by upregulated secretion of cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of cruzipain and chagasin are variable, but the molar ratios are fairly stable (∼50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild- type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild- type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells. [ABSTRACT FROM AUTHOR]