Endocytic Rabs Are Recruited to the Trypanosoma cruzi Parasitophorous Vacuole and Contribute to the Process of Infection in Non-professional Phagocytic Cells

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.

Keywords: Rab proteins; T. cruzi parasitophorous vacuole; Trypanosoma cruzi; endocytosis; host cell infection.

Figure 1

Kinetics of the recruitment of VAMP3 and VAMP7 on the T. cruzi parasitophorous vacuole. (A,B) CHO cells overexpressing GFP-VAMP3 or GFP-VAMP7 were infected with TCT (MOI 20) for 15 min or 6 h, respectively. The parasites were detected by indirect immunofluorescence using a specific antibody anti-T. cruzi followed by a secondary antibody labeled with Cy3 (red). The DNA of nuclei and kinetoplasts were detected with Hoechst (blue). Confocal images are representative for each condition and the magnifications are delimited in the original photo. Scale bar: 10 μm. (C) The percentage of the recruitment of VAMPs to TcPV were quantified from the images obtained by confocal microscopy at the indicated times after infection from 15 min to 18 h. Data are representative of three independent experiments.

Figure 2

Recruitment kinetics of Rab proteins to T. cruzi parasitophorous vacuole. (A) Recruitment of Rab to TcPV were examined at the indicated time–points. CHO cells overexpressing GFP-Rabs were infected for 15 min (15 mpi), 1, 3, 6, or 12 h (hpi) with TCT (MOI 20). The parasites were detected by indirect immunofluorescence using a specific antibody anti-T. cruzi followed by a secondary antibody labeled with Cy3 (red). The DNA of nuclei and kinetoplasts were detected with Hoechst (blue). Images are representative for each condition and the magnifications are delimited in the original photo. Scale bar: 10 μm. (B) Kinetic graphs represent the recruitment of GFP-Rabs to TcPV. Data are representative of two independent experiments. (***P < 0.001, **P < 0.01, *P < 0.05, two-way ANOVA and Bonferroni's multiple comparison test).

Figure 3

Effect of dominant-negative Rab-proteins on T. cruzi infection. (A) CHO cells overexpressing GFP-Rabs wild type (WT) or dominant-negative mutants were infected with TCT (MOI 20) for 1 h. After fixation, the parasites were detected by indirect immunofluorescence using a specific antibody anti-T. cruzi followed by a secondary antibody labeled with Cy3 (red). The DNA of nuclei and kinetoplasts were detected with Hoechst. Arrowheads indicate the localization of parasites. Scale bar: 10 μm. (B) Bar graph represents the percentage of infected cells at 1-h post-infection. For this experiment at least 120 cells were quantified. Data are representative of two independent experiments. (***P < 0.001, **P < 0.01, *P < 0.05, one-way ANOVA and Bonferroni's multiple comparison test). (C) Bar Graph represents the percentage of cells that contained 1, 2, or 3 or more parasites/cell at 1-h post-infection. For this experiment at least 120 cells were quantified. Data are representative of two independent experiments. (**P < 0.01, one-way ANOVA and Bonferroni's multiple comparison test).