CONICET | Buscador de Institutos y Recursos Humanos

Among the numerous isoforms of α-tubulin found in the differentmicrotubular structures of Trypanosoma cruzi, acetylated α-tubulinis one of the most abundant. In other organisms, one of the enzymesresponsible for this modification is the α-tubulin acetyltransferase,or αTAT.We have identified a coding sequence for the putative αTAT in thegenome of T. cruzi, established lines that overexpress the proteinof interest in an inducible manner, using the pTcINDEX-GW vectorand characterized the mutant parasites phenotypically. As a first approach,we evaluated the overexpression of the αTAT in the mutantsby western blot analysis (using anti-α-tubulin and anti-acetylatedα-tubulin as primary antibodies), showing an increase in the levelsof tubulin acetylation when αTAT is overexpressed. Besides, otherexperiments indicated that the protein localizes mainly in the cytoskeletonand flagellum of T. cruzi. Subsequently, we studied thegrowth of the mutant epimastigotes and a remarkable reduction wasobserved as a result of an increased expression of αTAT. Furthermore,we studied the morphology of the mutants by phase contrastmicroscopy and immunofluorescence analysis. The overexpressionof αTAT led to an abnormal morphology in the parasites; aberrantnuclei and parasites containing more than one flagellum were observed.In addition, a higher concentration of the protein was detectedmainly in the perinuclear region. Finally, we evaluated thephenotypic effect generated by orizalyn, a microtubule depolymerizingdrug. Wild-type parasites lose their normal morphology afterthe treatment in a dose-dependent manner but differences in drugresistance were observed in the overexpressing parasites upon theinduction.These findings suggest that the mechanisms of tubulin modification?particularly acetylation- could influence the functional role ofthe microtubules, both in cell division and differentiation during theparasite?s life-cycle.