Contractile vacuole complex as a part of classic secretory pathway in Trypanosoma cruzi

CAMPETELLA O; CARLEVARO G; MUCCI J; BURGOS J

Mar del Plata, Buenos Aires

Congreso; Reunión anual de sociedades de biociencia-XXXI reunión anual de la sociedad argentina de protozoología; 2019

SAP

T. cruzi has GPI-anchored virulence factors, such as trans-sialidase (TS) and mucins which are transported to the membrane by a non-conventional transport involving the contractile vacuole complex (CVC), an organelle absent in mammals. Both proteins are only detectable in the CVC during the differentiation to the infective stage of trypomastigote. There is scarce information concerning the role of CVC on this novel transport pathway. To analyze whether CVC is part of the general membrane protein traffic or is only involved in the transport of membrane proteins anchored by GPI, we study the intracellular traffic of proteins with different membrane anchorage according to bioinformatics predictions (myristoyl and palmitoyl, direct interaction with lipids, and GPI). These are: Calpain, KMP, and TolT3, three flagellar proteins present in the tripomastigote stage of T. cruzi. Traffic signals predicted in silico were confirmed by treatment with PI-PLC or constructs of proteins with its anchor sites mutated. The analysis of intracellular traffic was performed in the intermediate stage during the differentiation of amastigotes to trypomastigotes by confocal fluorescence microscopy. 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, we analyze the traffic of GFP fused to different traffic signals in order to become independent of the protein cores. For this, we use a tet-on inducible expression system, and we develop α- GST-Rab11 antibodies to label the CVC. We observed that both TolT3 and KMP colocalize with the CVC marker, while Calpain does not. Then, we observed the same with the GFP fused to the different traffic signals. Thus, our results indicate that CVC is not part of a general traffic route of proteins to the cell surface, but rather participates in the traffic of proteins that enter the classical secretory pathway.