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Abstract
Trypanosoma cruzi is the etiologic agent of Chagas disease. It contains a Contractile Vacuole Complex (CVC) that plays a vital role in the regulation of its cell volume and in its responses to osmotic stresses in all its life cycle stages. It is peculiar that though, T.cruzi is not a free-living organism it has a CVC; thus suggesting that the CVC could have functions beyond just osmoregulation as occurs in some other protists; where the CVC is involved in regulating calcium homeostasis and in the transfer of proteins to the surface. Besides, the approach of combined proteomic and bioinformatics analyses identified proteins localized to the CVC, several of them having trafficking roles, and implying to a potential novel role of the CVC.Here we used a combination of genetic and biochemical approaches to establish the contribution of the CVC as a trafficking hub. T. cruzi relies on protein secretion of glycosylphosphatidylinositol (GPI)-anchored surface proteins for invasion of host cells and establishment of infection. In this study we show that the CVC acts as a trafficking intermediate before GPI-anchored proteins reach the cell surface. Additionally we also identify CVC-located TcRab11 as a regulator of protein transport of GPI-anchored transsialidase to the plasma membrane, a process essential for the establishment of infection. Demonstration of the role of TcTS in infection has been previously difficult given the large number of genes encoding for this protein distributed through the genome of the parasite. We also studied the role of another CVC-located Rab. Rab32 is located in lysosome-related organelles (LRO) and since acidocalcisomes are LROs we investigated whether TcRab32 is needed for the structure and function of acidocalcisomes. By constructing GDP-bound dominant negative mutants of TcRab32 we were able to show a defect in trafficking, which ultimately affects parasite infectivity. This study with TcRab32 provides the link between the acidocalcisome and the contractile vacuole complex as observed in T. cruzi and in some other protists like Chlamydomonas reinhardtii and Dictyostelium discoideum.Our results are consistent with a role of the CVC in regulating membrane traffic to maintain the function of the acidocalcisome as well as traffic to the plasma membrane of T. cruzi.