CONICET | Buscador de Institutos y Recursos Humanos

Calcium (Ca2+) signaling modulates a plethora of diverse biological processes in the eukaryotic cell. To survive, the pathogen Typanosoma cruzi completes its life cycle passing from insect to vertebrate hosts and vice versa, as well as differentiating from extra or intracellular proliferative stages to cell cycle arrested forms. In such complex scenario, it is expected that Ca2+ signaling and homeostasis play pivotal roles regulating key processes such as proliferation and infection. Regarding this, experimental evidence has pointed out the importance of Ca2+ both in the parasite and host cells. In T. cruzi, it has been showed that the cytosolic Ca2+ ([Ca2+]c) increases in the differentiation from epimastigotes to metacyclic trypomastigotes. During the invasion into mammalian cells, the [Ca2+]c in trypomastigotes transiently augments as a consequence of parasite adhesion to the outer membrane of the host cell. In the last years, several channels involved in calcium flux have been partially characterized in T. cruzi, but the mechanisms decoding Ca2+ signals are not fully identified nor completely understood. Even more, the parasite genome codifies for several hypothetical Ca2+-binding proteins, which do not have homologous counterpart in the mammalian host, that remain uncharacterized. By carrying out a multi-step strategy search in the database tritrypdb.org, we selected one of these hypothetical Ca2+-binding proteins with experimental evidence of expression to investigate its role in T. cruzi in vitro proliferation, differentiation and host cell invasion. This 102-amino acid hypothetical protein, hereafter named TcCAL1, exhibits two putative EF-hand motifs for calcium binding and localizes along the cell body of the parasite in the main developmental forms. In order to infer the function of TcCAL1, we analyzed the effect of its over-expression in two different strains of T. cruzi epimastigotes, CL and Y (typified as TcI and TcII lineages, respectively). For this, we generated transgenic cultures from both strains overexpressing TcCAL1 fused to a 6X histidine tag (TcCAL1-6XHis). Parasites transfected by electroporation with the empty expression vector were used as control culture. We found that the overexpression of TcCAL1-6XHis did not affect proliferation for CL or Y epimastigote forms in the exponential phase of growth, compared with their respective controls. We then performed in vitro metacyclogenesis assays to investigate whether the overexpression of TcCAL1-6XHis affected the differentiation from epimastigotes into infective forms. Results showed that metacyclogenesis was impaired in TcCAL1-6XHis-overexpressing parasites from both strains. In fact, the percentages of metacyclic trypomastigotes from CL or Y strains overexpressing TcCAL1-6XHis significantly decreased 70 or 40%, respectively, compared to control cultures. Then, we evaluated the virulence of transgenic metacyclic trypomastigotes from the Y strain in mammalian cell invasion assays. We found that the infection percentages were significantly higher in Vero cells incubated with TcCAL1-6XHis-overexpressing parasites than controls (41.2±6.23 versus 18.9±5.34%, respectively), as well as the number of intracellular amastigotes (1398±48 versus 926±124 parasites/100 host cells). In addition, adhesion assays showed that percentages of Vero cells with adhered metacyclic trypomastigotes were significantly higher in samples incubated with TcCAL1-6XHis-overexpressing parasites compared with controls (76.3±14 versus 44.8±8.5%, respectively). Finally, preliminary results showed that the differentiation rates from metacyclic trypomastigotes to axenic amastigotes were not affected by the overexpression of TcCAL1x6His. In summary, we showed that the overexpression of TcCAL1 in T. cruzi impairs the metacyclogenesis of epimastigotes but enhances the infectivity of metacyclic tripomastigotes to invade Vero cells. Our finding suggests that TcCAL1 could exert its function sequestering intracellular calcium by its EF-hands motifs (impairing metacyclogenesis) and/or due to an unknown activity which could be amplified upon Ca2+ binding (promoting cell invasion). This work corroborates the importance to study the hypothetical proteins of unknown function in pathogen parasites.