A Rab32-dependent pathway contributes to Salmonella typhi host restriction

Abstract

Unlike other Salmonellae, the intracellular bacterial human pathogen Salmonella Typhi exhibits strict host specificity. The molecular bases for this restriction are unknown. Here we found that the expression of a single type III secretion system effector protein from broad-host Salmonella Typhimurium allowed Salmonella Typhi to survive and replicate within macrophages and tissues from mice, a nonpermissive host. This effector proteolytically targeted Rab32, which controls traffic to lysosome-related organelles in conjunction with components of the biogenesis of lysosome-related organelle complexes (BLOCs). RNA interference-mediated depletion of Rab32 or of an essential component of a BLOC complex was sufficient to allow S. Typhi to survive within mouse macrophages. Furthermore, S. Typhi was able to survive in macrophages from mice defective in BLOC components.

Fig. 1. GtgE expression limits host-cell restriction

(A) Survival of S. Typhi expressing GtgE in bone-marrow-derived mouse macrophages (BMDM). Macrophages were infected with S. Typhi (Ty), S. Typhi expressing GtgE (Ty GtgE), or S. Typhimurium (Tm). Cells were lysed at the indicated time points and c. f. u. enumerated. Values are means ± SEM of three independent measurements. p values (for the difference relative to values obtained from cells infected with S. Typhi) were determined by the Student’s t test: *<0.05; **<0.01; ***<0.001. (B) and (C) C. f. u. recovered from the spleens of C57BL/6 Nramp^+/+ mice infected with S. Typhi (wt) or S. Typhi expressing GtgE (GtgE) 4 days after intraperitoneal (B) or oral (C) inoculation. Horizontal bars indicate the means. The p value was determined by the Wilcoxon rank sum test.

Fig. 2. Rab32 and Rab38 are targets of the GtgE protease activity

(A) GtgE cleaves Rab32 and Rab38 in cultured cells. COS-1 cells were co-transfected with plasmids expressing the indicated GFP or RFP-tagged Rab GTPases and wild-type GtgE (wt) or a GtgE catalytic mutant (GtgE^H151A) (cat). Twenty-four hours after transfection, cells were lysed and samples were analyzed by Western blotting using a rabbit anti-GFP antibody or a rabbit anti-RFP antibody. (B) GtgE cleaves Rab32 and Rab38 in vitro. Purified MBP-tagged Rab32 or MBP-tagged Rab38 were incubated with MBP-tagged GtgE, separated by SDS-PAGE and stained with Coomassie. (C) GtgE targets Rab32 and Rab38 during Salmonella infection. COS-1 cells expressing GFP-Rab29, YFP-Rab32, YFP-Rab38 or RFP-Rab23 were infected with S. Typhi (Ty), or S. Typhimurium (Tm). Two and a half hour after infection cells were lysed and samples were analyzed by Western blotting using a rabbit anti-GFP antibody or a rabbit anti-RFP antibody. (D) Phylogenetic tree of the human Rab and Rab-like GTPases. The locations of Rab29, Rab32 and Rab38 within the tree are indicated in red.

Fig. 3. Rab32 and Rab38 are recruited to the Salmonella Typhi-containing vacuole

Henle-407 cells (A and B) or mouse primary BMDM (C) expressing YFP- or CFP-tagged Rab32 or Rab38 (green) were infected with S. Typhi (A and C), S. Paratyphi (B and C), or S. Typhimurium (B and C) expressing mCherry (red) and imaged at the indicated times (A) or 2 hours (B and C) after infection. The images shown represent maximum intensity projections of Z-stacks. Bars, 10 μm.

Fig. 4. Rab32 and BLOC-3 are required for S. Typhi host-cell restriction

(A) Intracellular survival of S. Typhi in BMDM depleted of Rab32 and Rab38. BMDM from mice were transfected with a non-targeting siRNA smart pool (nt) or siRNA smart pools targeting Rab32 or Rab38. Three days after transfection cells were infected with S. Typhi, lysed at the indicated time points after infection and c. f. u. were enumerated. Values are means ± SEM of three independent measurements. (B) Schematic representation of the comparison between the maturation of the Salmonella-containing vacuole and melanosomes (an example of a lysosome-related organelle). (C) Intracellular survival of S. Typhi in BMDM depleted of BLOCs. BMDM from mice were nucleoporated with a non-targeting siRNA smart pool (nt) or specific siRNA smart pools targeting HPS7 (subunit of BLOC-1), HPS6 (subunit of BLOC-2) or HPS4 (subunit of BLOC-3). Three days after transfection, cells were infected with S. Typhi, lysed at the indicated time points and c. f. u. were enumerated. Values are means ± SEM of three independent experiments. (D) Intracellular survival of S. Typhi in BMDM defective for BLOC-1, -2, and -3. BMDM cells from mice simultaneously defective in BLOC-1, 2, and -3 (pa/co/le) were infected with S. Typhi, lysed at the indicated time points after infection and c. f. u. were enumerated. Values are means ± SEM of three independent measurements. (E) Alignment of human and mouse Rab32 shows sequence variation in the N-terminal and C-terminal regions. (F) Heat-map of the degrees of identity of the human Rab GTPases with the orthologs in three mammalian species: Pan troglodytes, Sus scrofa, Mus musculus. Black represents 100% identity, white represents 75% identity. p values were determined by the Student’s t test: *<0.05; **<0.01; ***<0.001.