EXPERIMENTAL APPROACHES FOR THE FUNCTIONAL STUDY OF A PUTATIVE CALCIUM BINDING PROTEIN IN TRYPANOSOMA CRUZI

POTENZA M; OSUNA CARRILLO A ; WEHRENDT D. P; TELLEZ- IÑÓN M. T

Congreso; XXVII Reunión Anual de la Sociedad Argentina de Protozoología; 2015

Data from genome annotation and mass spectrometry projects of pathogens has allowed the identification of novel proteins, for which their putative functions cannot be predicted by sequence similarity. Trypanosoma cruzi, the etiological agent of Chagas disease, has a complex life cycle in which cell differentiation and invasion are tightly coordinated. In this parasite, calcium and calcium binding proteins play important roles in the processes of mammalian cell infection and survival. Using a rational search engine, we selected an uncharacterized gene, named TcCALI which encodes a 103 amino acid protein with two putative calcium binding sites. Previously, we found that this protein is able to form disulphide bonds and has a putative palmitoylation domain. We also found TcCALI localizes along the cytoplasm in all differential stages of the parasite and several protein bands at higher molecular weight than expected are detected by western blot. In order to study TcCALI´s role on the parasite physiology, we searched for its associated components, to characterize the metabolic environment of this protein with unknown function. To this aim, two experimental approaches were addressed. First, protein extracts from T. cruzi were immunoprecipitated using anti-TcCALI antibodies and commercial kits consisting of recombinant Protein G covalently coupled to magnetic beads. The results showed that TcCALI associates to one or more components, which probably modify the TcCALI conformation. On the other hand, yeast two hybrid assays are undergoing with the objective to identify TcCALI binding partners from a cDNA library. For this, TcCALI and Lamin (negative control) were cloned separately in the shuttle vector pBMT116 and transfected to S. cereviseae (L40 strain). Selection of recombinant yeast was achieved plating transformants in minimal media. Future steps involve transfection of TcCALI expressing yeasts with a T. cruzi cDNA library cloned in the pVP16 vector.