Introduction Cysteine peptidases (CPs) belonging to the C1 family of the papain superfamily have been implicated in the control of growth, differentiation and survival of several pathogenic protozoa (for a review, see Sajid and McKerrow, 2002). In Trypanosoma cruzi, the causative agent of human Chagas’ disease, studies with synthetic irreversible CP inhibitors suggested that T. cruzi infectivity and intracellular growth depend on the activity of cruzipain, the main lysosomal CP (Meirelles et al., 1992; Harth et al., 1993; Engel et al., 1998a). Encoded by a large polymorphic gene family (Eakin et al., 1992; Campetella et al., 1992), the mature form of cruzipain has a central domain homologous to mammalian lysosomal CPs but this is linked to a unique 130-residue C-terminal extension with unknown function (for a review, see Cazzulo et al., 1997). Cloning and heterologous expression of a few polymorphic cruzipain-encoding genes revealed that some of these isoenzymes display significant differences in their substrate specificities and kinetic properties (Lima et al., 1994; Lima et al., 2001). The solution of the crystal structure of the catalytic domain (McGrath et al., 1995) paved the way for the development of new generations of irreversible synthetic inhibitors, some of which could protect mice from lethal T. cruzi infection (Engel et al., 1998b). Recent clues to cruzipain function have come from analyses of the Ca 2+ -dependent signalling pathways driving T. cruzi invasion of endothelial cells and cardiomyocytes (Scharfstein, 2000; Todorov et al., 2003). These studies revealed that parasite infectivity is increased by activation of G-protein-coupled kinin receptors (for a review, see Scharfstein, 2003). Of further interest, recent studies suggested that alternative signalling routes, also dependent on cruzipain-mediated proteolysis, drive T. cruzi invasion of human smooth-muscle cells (Aparicio et al., 2004). Although cruzipain has been extensively characterized at the biochemical (Serveau et al., 1996; Del Nery et al., 1997; Meldal et al., 1998; Serveau et al., 1999; Lima et al., 2001; Lima et al., 2002) and structural (McGrath et al., 1995; Gillmor et al., 1997) levels, little is known about the mechanisms 901 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. Key words: Trypanosome, Cysteine protease, Chagasin, Inhibitor, Infectivity Summary Chagasin, the endogenous cysteine-protease inhibitor of Trypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells Camila C. Santos, Celso Sant’Anna, Amanda Terres, Narcisa L. Cunha-e-Silva, Julio Scharfstein and Ana Paula C. de A. Lima* Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, C.C.S., Ilha do Fundão, Rio de Janeiro, 21949-900 RJ, Brazil *Author for correspondence (e-mail: anapaula@biof.ufrj.br) Accepted 8 December 2004 Journal of Cell Science 118, 901-915 Published by The Company of Biologists 2005 doi:10.1242/jcs.01677 Research Article Journal฀of฀Cell฀Science