Identification of proteins localized to the contractile vacuole of Trypanosoma cruzi
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Trypanosoma cruzi is the etiologic agent of Chagas disease and is able to survive in a wide range of environments as it progresses through its life cycle. Fluctuations in osmolarity occur within the gut of the vector and as the parasite moves from the insect gut to the bloodstream, acidic phagolysosomes, and host cell cytosol. Thus, the parasites have mechanisms to respond to both hypo-osmotic and hyper-osmotic stresses. The contractile vacuole complex is an osmoregulatory organelle, which controls intracellular water balance by accumulating excess water and expelling it from the cell. Although recent studies showed the contractile vacuole is involved in volume regulation in T. cruzi, how the contractile vacuole mediates this process is poorly understood. We will identify the proteins present in the contractile vacuole in order to clarify the mechanism. In a previous proteomic study done in the laboratory a number of proteins were identified in a fraction enriched for the contractile vacuole. To validate the proteomic data we chose to confirm the localization of proteins found in this fraction and with homology to those found also in the Paramecium tetraurelia, Dictyostelium discoideum and Acanthamoeba castellanii contractile vacuole complex. V-H+-ATPase, LvsA, drainin and Rab11 are known to regulate the contractile vacuole system and the pathways that control contractile vacuole discharge in Dictyostelium. Clathrin and AP180, major components of the cytoplasmic coat found on clathrin coated vesicles localize to the contractile vacuole and are required for the biogenesis of the contractile vacuole system in Dictyostelium. SNAREs are intracellular trafficking proteins that localize to the contractile vacuole and are involved in the function of the contractile vacuole in Paramecium. In order to study the localization of these proteins, we cloned six contractile vacuole protein-coding genes (V-H+-ATPase subunit B, V-H+-ATPase subunit a, SNARE2-1, SNARE2-2, AP180, and Disgorgin/Drainin) from T. cruzi. To localize contractile vacuole proteins, we generated stable cells overexpressing the contractile vacuole proteins tagged with green fluorescence protein (GFP). As a result, we show that the TcV-H+-ATPase subunit B, subunit a, and TcAP180 localize to the bladder of the contractile vacuole, two SNAREs, TcSNARE2.1 and TcSNARE2.2 localize to the spongiome of the contractile vacuole, and TcDisgorgin/Drainin is cytsolic and in the flagella pocket. Overall, we have demonstrated the presence of six new contractile vacuole proteins in T. cruzi using proteomic analysis and overexpression of the contractile vacuole proteins with GFP fusion in T. cruzi.