CONTRACTILE VACUOLE COMPLEX AS PART OF THE CLASSIC SECRETORY PATHWAY IN TRYPANOSOMA CRUZI

JUAN M. BURGOS; GIANNINA CARLEVARO; JUAN MUCCI; OSCAR CAMPETELLA

Congreso; XXXII Reunión Anual Sociedad Argentina de Protozoología; 2020

T. cruzi has glycosylphosphatidylinositol (GPI) anchored virulence factors, such as trans-sialidase (TS) and mucins which are transported to the membrane through a non-conventional pathway involving the contractile vacuole complex (CVC), an organelle absent in mammals. There is still scarce information concerning the role of CVC in the general transport of proteins to the surface. To analyze whether CVC is part of the global membrane protein traffic or is only involved in the transport of proteins anchored by GPI, we studied the intracellular traffic of proteins with different membrane anchorage according to bioinformatics predictions (myristoyl and palmitoyl, direct interaction with lipids, and GPI). Calpain, KMP and TolT3 were selected as model proteins, respectively, three flagellar proteins present in the trypomastigote stage of T. cruzi. Traffic signals predicted in silico were confirmed by treatment with PI-PLC or protein overexpression with their anchor sites mutated. The analysis of intracellular traffic was performed by confocal fluorescence microscopy in the intermediate stage during the differentiation of amastigotes to trypomastigoteswhen flagellar protein transport is increased. For this we obtained a population of transfected parasites that express a CVC protein fused to GFP (the GFP-GTPase Rab11) as a marker of this organelle. Finally, to become independent of the protein cores, the traffic of GFP fused to different traffic signals was analyzed. For this we used a tet-on inducible expression system and we developed anti-GST-Rab11 antibodies to label the CVC. We observed that both TolT3 and KMP colocalized with the CVC marker while Calpain did not. Same results were obtained with the GFP fused to the different traffic signals. Thus, our results indicate that CVC does not participate in a general route of protein traffic to the cell surface, but rather is part of the classic secretory pathway in T. cruzi.