Abstract
Trypanosoma cruzi is the parasite causative of Chagas disease, a highly disseminated illness endemic in Latin-American countries. T. cruzi has a complex life cycle that involves mammalian hosts and insect vectors both of which exhibits different parasitic forms. Trypomastigotes are the infective forms capable to invade several types of host cells from mammals. T. cruzi infection process comprises two sequential steps, the formation and the maturation of the Trypanosoma cruzi parasitophorous vacuole. Host Rab GTPases are proteins that control the intracellular vesicular traffic by regulating budding, transport, docking, and tethering of vesicles. From over 70 Rab GTPases identified in mammalian cells only two, Rab5 and Rab7 have been found in the T. cruzi vacuole to date. In this work, we have characterized the role of the endocytic, recycling, and secretory routes in the T. cruzi infection process in CHO cells, by studying the most representative Rabs of these pathways. We found that endocytic Rabs are selectively recruited to the vacuole of T. cruzi, among them Rab22a, Rab5, and Rab21 right away after the infection followed by Rab7 and Rab39a at later times. However, neither recycling nor secretory Rabs were present in the vacuole membrane at the times studied. Interestingly loss of function of endocytic Rabs by the use of their dominant-negative mutant forms significantly decreases T. cruzi infection. These data highlight the contribution of these proteins and the endosomal route in the process of T. cruzi infection.
Highlights
The Ras-associated binding proteins, Rabs, are small GTP–binding proteins that control the budding, transport, docking, and tethering of vesicles from a donor to an acceptor compartment
In a previous work we studied the recruitment of the SNARE proteins VAMP3 and VAMP7 to the parasitophorous vacuole of T. cruzi CL Brener strain at different times after infection
In this work we used the T. cruzi Y strain, we studied the recruitment of these SNAREs and confirmed their presence in the vacuole membrane (Figures 1A,B)
Summary
The Ras-associated binding proteins, Rabs, are small GTP–binding proteins that control the budding, transport, docking, and tethering of vesicles from a donor to an acceptor compartment. The class C VPS/HOPS complex (Rieder and Emr, 1997; Seals et al, 2000), is a Rab effector that promotes the arrival of Rab and the sorting of endocytosed ligands from early to late endosomes which, in turn, will mature into lysosomes, the degradative compartment of the cell (Rink et al, 2005). Rab39a is a poorly characterized Rab located in endocytic compartments that regulates transport and fusion of vesicles from the Golgi complex to LE/multivesicular bodies (Chen et al, 2003; Gambarte Tudela et al, 2019). Due to antibodies against Rab proteins have low sensitivity, the most important tool used to study these proteins has been the expression of mutant proteins defective in the binding (dominant-negative mutants) or in the hydrolysis (dominant-positive mutants) of GTP. These mutants fused to GFP have been widely used to localize and to describe the function of a particular Rab in a particular traffic (Tisdale et al, 1992; Zerial and Stenmark, 1993)
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