Yersinia pestis Requires Host Rab1b for Survival in Macrophages

Abstract

Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

Fig 1. Rab1b knockdown inhibits the survival of Y. pestis within macrophages.

RAW264.7 macrophages were reverse transfected with Rab1a, Rab1b, scrambled (Scr), or Copβ1 siRNA. 48 h after transfection cells were infected with Y. pestis (MOI 10). (A) Percent survival of intracellular CO92 pCD1^(-) Lux_PtolC in Rab1a or Rab1b siRNA treated macrophages as compared to Scr siRNA treated macrophages. (B) Bioluminescence of intracellular bacteria from macrophages infected for 10 h with Y. pestis CO92 pCD1^(-) Lux_PtolC. (C) Conventional enumeration of intracellular bacteria from macrophages infected for 10 h with Y. pestis CO92 pCD1^(-) Lux_PtolC. (D) Bioluminescence of intracellular bacteria from macrophages infected for 2 h with Y. pestis CO92 pCD1^(-) Lux_PtolC. (E) Bioluminescence of intracellular bacteria from macrophages infected for 10 h with Y. pestis KIM D-19 Lux_PtolC. (F) Conventional enumeration of intracellular bacteria from macrophages infected for 10 h with Y. pestis KIM D-19 Lux_PtolC. (G) Bioluminescence of intracellular bacteria from macrophages infected for 2 h with Y. pestis KIM D-19 Lux_PtolC. The limit of detection for conventional enumeration is denoted by the dotted line. RLU = Relative Light Units; CFU = Colony Forming Units. *** = p<0.001, **** = p<0.0001.

Fig 2. Rab1b knockdown does not impact Y. pestis invasion of macrophages.

RAW264.7 macrophages were reverse transfected with Rab1b, scrambled (Scr), or Copβ1 siRNA. 48 h after transfection cells were infected with Y. pestis CO92 pCD1^(-)pGEN-P _EM7::DsRED (MOI 7.5). 20 or 80 min post-infection cells and bacteria were fixed with paraformaldehyde and extracellular bacteria were stained by indirect immunofluorescence with anti-Y. pestis antibody. (A) Representative image showing differential staining of intracellular (red) and extracellular (green or yellow) bacteria. Scale bar is 5μm. Asterisk denotes intracellular Y. pestis. (B and C) Percentage of intracellular bacteria calculated at 20 and 80 min post-infection, respectively. ** = p<0.01, ns = not significant.

Fig 3. Rab1b knockdown alters YCV acidification.

RAW264.7 macrophage cells were reverse transfected with scrambled (Scr), Rab1b or Copβ1 siRNA. 48 h after transfection cells were incubated with Lysotracker Red DND-99 for 1 h and then infected with live or paraformaldehyde-killed Y. pestis CO92 pCD1^(-) pGEN222 expressing EGFP (MOI 7.5). Colocalization of Lysotracker Red DND-99 and Y. pestis CO92 pCD1^(-) pGEN222 was determined by confocal microscopy. (A) Representative images showing colocalization of Lysotracker Red DND-99 and Y. pestis. Scale bar is 5μm. (B) Percent of YCVs that colocalized with Lysotracker Red DND-99 at 20 min post-infection. (C) Percent of YCVs that colocalized with Lysotracker Red DND-99 at 80 min post-infection. ** = p<0.01, *** = p<0.001.

Fig 4. Rab1b knockdown increases YCV association with Lamp1.

RAW264.7 macrophage cells were reverse transfected with either scrambled (Scr) or Rab1b siRNA. 48 h after transfection cells were infected with live or paraformaldehyde-killed Y. pestis CO92 pCD1^(-) pGEN-EM7::DsRED (MOI 3). Cells were stained for Lamp1 and colocalization was determined by confocal microscopy. (A) Representative images showing bacterial colocalization with Lamp1 at 20 min post-infection. Colocalization channel was defined using Imaris software. Asterisks denote bacteria not colocalized with Lamp1; arrowheads denote bacteria colocalized with Lamp1. Scale bar is 5μm. (B) Percent of YCVs that colocalized with Lamp1 at 20 min post-infection. (C) Percent of YCVs that colocalized with Lamp1 at 80 min post-infection. ** = p<0.01, *** = p<0.001.

Fig 5. Rab1b knockdown does not affect YCV association with LC3.

RAW264.7 macrophage cells were reverse transfected with either scrambled (Scr) or Rab1b siRNA. 48 h after transfection cells were infected with live or paraformaldehyde killed Y. pestis CO92 pCD1^(-) pGEN-P _EM7::DsRED (MOI 7.5). Cells were stained for LC3 and colocalization was determined by confocal microscopy. (A) Representative images showing bacterial colocalization with LC3 at 20 min post infection. The colocalization channel was defined using Imaris software. Asterisks denote bacteria not colocalized with LC3; arrowheads denote bacteria colocalized with LC3. Scale bar is 5μm. (B) Percent of YCVs that colocalized with LC3 at 20 min post-infection. (C) Percent of YCVs that colocalized with LC3 at 80 min post-infection. ns = not significant.

Fig 6. Rab1b is recruited to the YCV.

RAW264.7 macrophages were transiently transfected with pEGFP-Rab1B(CA). 24 h after transfection cells were infected with either live or paraformaldehyde killed Y. pestis pMCherry (MOI 7.5) or E. coli pMCherry (MOI 20). Colocalization of EGFP-Rab1b(CA) and bacteria was determined by confocal microscopy. (A) Representative images showing bacterial colocalization with EGFP-Rab1b(CA). Colocalization channel was defined using Imaris software. Asterisks denote bacteria not colocalized with EGFP-Rab1b(CA); arrowheads denote bacteria colocalized with EGFP-Rab1b(CA); arrows denote bacteria in untransfected cells. Scale bar is 5μm. (B and C) Percent of bacteria colocalized with EGFP-Rab1B(CA) at 20 and 80 min post-infection. * = p<0.05; ** = p<0.01; *** = p<0.001.

Fig 7. Inhibition of the secretory pathway does not inhibit Y. pestis intracellular survival.

RAW264.7 macrophages were treated with 0, 0.2 or 10 μM BFA prior to infection with Y. pestis CO92 pCD1^(-) Lux_PtolC (MOI 10). Extracellular bacteria were killed with gentamicin and intracellular bacterial numbers were monitored at (A) 2 h and (B) 10 h post-infection by bioluminescence. To determine if BFA treatment impacted the ability of Y. pestis to inhibit YCV acidification, macrophages treated with 10 μM BFA were incubated with Lysotracker Red DND-99 prior to infection with live or paraformaldehyde killed Y. pestis CO92 pCD1^(-) pGEN222 (MOI 3). Bacterial Colocalization with Lysotracker Red DND-99 was determined by confocal microscopy at (C) 20 and (D) 80 min post-infection. ns = not significant.

Fig 8. Knockdown of Rab1b increases L. pneumophila LCV acidification.

RAW264.7 macrophage cells were reverse transfected with either scrambled (Scr) or Rab1b siRNA. 48 h after transfection cells were incubated with Lysotracker Red DND-99 for 1 h, and infected with L. pneumophila pMIP-GFP (MOI 10). Coverslips were fixed and colocalization of Lysotracker was determined by confocal microscopy. (A) Representative images showing colocalization of Lysotracker with L. pneumophila. Scale bar is 5μm. (B) Percent of LCVs that colocalized with Lysotracker at 20 min post-infection. (C) Percent of LCVs that colocalized with Lysotracker Red DND-99 at 80 min post-infection. ** = p<0.05, *** = p<0.001.