CONICET | Buscador de Institutos y Recursos Humanos

Understanding the role of calcium-related genes in Trypanosoma cruzi biology, the causative agent of Chagas disease, could broaden the scope in research of new drug targets against this parasite. Evidences have shown that Ca2+ may play an important role in T. cruzi differentiation and mammalian infection, processes that assure parasite survival and life cycle continuity. In order to define new specific Ca2+-mediated signal transduction pathways in trypanosomatids, we start to study some uncharacterized gene products that present putative calcium binding domains. For this, we performed a search at TritrypDB web site, a database containing the annotation and analysis of some pathogenic trypanosomatid genome projects. As a search engine, we use the terms ?hypothetical protein?, ?calcium ion binding?, ?proteome evidence? and ?phosphorylation proteome evidence?. As a result, four proteins were found and one of them was selected for further study. This 103 amino acid sequence, named TcCalI, presents several EF-Hand motifs and one palmitoylation site as predicted by bioinformatics tools. We cloned and expressed TcCalI in bacteria and then raised antibodies against the purified recombinant protein. Using SDS-polyacrylamide gel electrophoresis followed by the addition of dithiothreitol, we observed homo-dimerization of TcCalI via inter-molecular disulphide bond formation. By western blots, we found that TcCalI is expressed in all the three stages of T. cruzi, showing bands at the predicted molecular weight as well as two higher bands. We also found that TcCalI localizes in the cytoplasm and along the flagellum in epimastigote and trypomastigote forms, as could be evidenced by immunofluorescence microscopy. To better identify and define the function of TcCall, Ca+2 binding, immune-precipitation and yeast two hybrid experiments will be performed.