CONICET | Buscador de Institutos y Recursos Humanos

Calcium homeostasis is implicated in essential processes of T. cruzi life cycle. Although several calcium binding proteins have been identified in T. cruzi genome, their role in Ca2+ signaling remain still unknown. TcCAL1 is a hypothetical protein identified through proteome analysis of T. cruzi, which has two EF-Hand domains involved in Ca2+ binding. The hypothesis to be tested in this study was that the overexpression of TcCAL1 could trigger alterations in the intracellular calcium homeostasis leading an effect on epimastigote proliferation. For this purpose, the gene encoding tccal1 was amplified by PCR and cloned fussed to a 6x histidine tag into the pTREX expression vector. The recombinant plasmid pTREX/tccal1x6His was used to transfect epimastigotes from T. cruzi Y or CL Brener strains. Selection of transgenic cultures was carried out growing parasites in presence of increasing concentrations of Geneticin. Overexpression of TcCAL1x6His fusion protein was confirmed through western blot and affinity chromatography. Growth curves of recombinant cultures from Y or CL Brener strains were performed in triplicate, counting the parasites in a Neubauer chamber. Parasite cultures carrying the empty vector pTREX were used as controls. Data obtained were processed using GraphPad Prism 6.0 Software and subjected to statistical analysis by two-way ANOVA test. The preliminary results shown that the overexpression of TcCAL1x6His affects the growth curve of CL Brenner strain at the stationary phase but the proliferation Y strain is not affected. Additional experiments are being carrying out to validate these results and to study the effect of TcCAL1 overexpression on T. cruzi differentiation. Future assays including parasites carrying the tccal1 gene silenced will contribute to validate our hypothesis and to reveal the function of TcCAL1 in calcium homeostasis of T. cruzi.