VARP and Rab9 Are Dispensable for the Rab32/BLOC-3 Dependent Salmonella Killing

Abstract

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever, a disease that kills an estimated 200,000 people annually. Previously, we discovered an antimicrobial pathway dependent on Rab32 and BLOC-3 (BRAM) that is critical to kill S. Typhi in murine macrophages. The BLOC-3 complex is comprised of the two sub-units HPS1 and HPS4 and exhibits guanine-nucleotide exchange factor (GEF) activity to Rab32. In melanocytes, Rab9 has been shown to interact with HPS4 and RUTBC1, a Rab32 GTPase activating (GAP) protein, and regulate the Rab32-mediated melanosome biogenesis. Intriguingly, Rab9-deficient melanocytes exhibit hypopigmentation, a similar phenotype to Rab32 or BLOC-3 deficient melanocytes. Additionally, VPS9-ankyrin-repeat-protein (VARP) has been shown to regulate melanocytic enzyme trafficking into the melanosomes through interaction with Rab32. Although Rab32, Rab9 and VARP are a part of melanogenesis in melanocytes, whether Rab9 and VARP are required for the BRAM mediated killing in macrophages is currently unknown. Here we showed that HPS4 is recruited to the Salmonella-containing vacuoles (SCV) and over-expression of BLOC-3 significantly increased Rab32-positive bacteria vacuoles. We found that SCV acquire Rab9, however over-expressing Rab9 did not change HPS4 localization on bacteria vacuoles. Importantly, we used shRNA to knock-down Rab9 and VARP in macrophages and showed that these proteins are dispensable for Rab32 recruitment to the SCV. Furthermore, we assessed the survival of S. Typhimurium in macrophages deficient for Rab9 or VARP and demonstrated that these proteins are not essential for BRAM pathway-dependent killing.

Keywords: BLOC-3; Rab32; Rab9; Salmonella; VPS9-ankyrin-repeat-protein; macrophages.

Figure 1

BLOC-3 sub-unit HPS4 is recruited to the Salmonella-containing vacuoles (SCV). (A) HeLa cells were infected with mCherry S. Typhimurium ΔgtgEΔsopD2 (ΔΔ) or WT for 2.5 h, fixed and stained with monoclonal anti-HPS4 antibody (green). (B) The percentage of S. Typhimurium in HPS4-positive vacuoles were analyzed by immunofluorescence and the ± standard deviation of three independent experiments are shown. At least 100 bacteria were counted in each experiment. (C) HeLa cells either over-expressing BLOC-3 complex or empty plasmid (control) were infected with S. Typhimurium ΔgtgEΔsopD2, fixed at 2.5-h post-infection and co-stained with anti-HPS4 (green) and anti-Rab32 (gray) antibodies. Images were analyzed using fluorescence microscopy. (D) The percentage of bacterium in HPS4 or Rab32 positive vacuoles were analyzed by immunofluorescence and the ± standard deviation of three independent experiments are shown. (Student’s t test; *p < 0.05, NS, Non-significant), S. Tm, Salmonella Typhimurium; Scale bar: 5μm.

Figure 2

Rab9 does not regulate Rab32 recruitment to the Salmonella-containing vacuoles (SCV) and its function is not required for Rab32/BLOC-3 dependent killing. (A) GFP-Rab9 transfected HeLa cells were infected with mCherry S. Typhimurium ΔgtgEΔsopD2 (ΔΔ) or WT for 2.5 h, fixed and images were acquired using fluorescent microscopy. (B) The percentage of S. Typhimurium in Rab9-positive vacuoles at 2.5 h post-infection is shown with ± standard-deviation of three independent experiments. (C) HeLa cells transfected with either GFP or GFP-Rab9 were infected with mCherry S. Typhimurium WT for 2.5 h. Cells were then fixed, stained with monoclonal anti-HPS4 (gray) antibody and analyzed by fluorescence microscopy. (D) The percentage of S. Typhimurium in HPS4-positive vacuoles at 2.5 h post-infection is shown with ± standard-deviation of three independent experiments. (E) iBMDM cells knocked down for Rab9 (Rab9 KD) and control (Scrmbl) were infected with mCherry S. Typhimurium ΔgtgEΔsopD2 or WT for 2.5 h, fixed and stained with anti-Rab32 (green). Images were acquired using fluorescent microscopy (F) The percentage of S. Typhimurium in Rab32-positive vacuoles is shown with the ± standard-deviation from three independent experiment. (G) iBMDM cells depleted for Rab9 (Rab9 KD) and control cells (Scrmbl) were infected with S. Typhimurium ΔgtgEΔsopD2 or WT, lysed at the indicated time points and colony-forming units were calculated. Values are presented as fold change compared to the initially internalized (1.5 hpi). Error bars indicate standard-deviation of five independent experiments. For the non-normalized values see Figure S2 . (Student’s t test; *p < 0.05, **p < 0.01) S. Tm, Salmonella Typhimurium; KD, Knock-down; CFUs, Colony-forming units; Scale bar: 5μm; NS, non statistically different.

Figure 3

VPS9-ankyrin-repeat-protein (VARP) is not required for Rab32 trafficking to Salmonella-containing vacuoles (SCV) and its role in Salmonella killing is dispensable. (A) BMDM cells transduced with control (Scramble) or VARP shRNA (VARP KD) were infected with mCherry S. Typhimurium ΔgtgEΔsopD2 (ΔΔ) or WT for 2.5 h, fixed and stained with anti-Rab32 (green). Images were acquired using fluorescent microscopy. (B) The percentage of S. Typhimurium in Rab32-positive vacuoles were quantified in scramble and VARP KD cells and the ± standard-deviation of three independent experiments are shown. (C) BMDM cells depleted for VARP were infected with S. Typhimurium ΔΔ or WT, lysed at the indicated time points and colony-forming units were calculated. Values are standard-deviation of CFUs at each time points from three independent experiments. S. Tm, Salmonella Typhimurium; KD, Knock-down; CFUs, Colony-forming units; Scale bar: 5μm. (Student’s test; **p < 0.01, ***p < 0.001, ****p < 0.0001, NS, non statistically different).