Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

Abstract

Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.IMPORTANCEYersinia pestis can infect and survive within macrophages. However, the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. To provide a better understanding of these mechanisms, we used an RNAi approach to identify host factors required for intracellular Y. pestis survival. This approach revealed that the host endocytic recycling pathway is essential for Y. pestis to avoid clearance by the macrophage. We further demonstrate that Y. pestis remodels the phagosome to resemble a recycling endosome, allowing the bacterium to avoid the normal phagolysosomal maturation pathway. Moreover, we show that infection with Y. pestis disrupts normal recycling in the macrophage and that disruption is required for bacterial replication. These findings provide the first evidence that Y. pestis targets the host endocytic recycling pathway in order to evade killing by macrophages.

Keywords: Rab GTPases; Yersinia pestis; endosome recycling; intracellular survival; plague.

FIG 1

RNAi-based assay to identify host factors required for intracellular survival of Y. pestis. To determine whether reproducible RNAi could be achieved in RAW264.7 macrophages, cells were reverse transfected with siRNAs targeting indicated genes. (A and B) Forty-eight hours posttransfection, cells (n = 3) were harvested for RNA isolation and qRT-PCR (data represent the level of gene expression compared to the level for the scrambled siRNA control) (A) or protein isolation for Western blot analysis (β-actin was used as a loading control) (B). α-GAPDH, anti-GAPDH antibody; α-βactin, anti-βactin antibody. To demonstrate that the Y. pestis CO92 pCD1^(-) Lux_PtolC bioreporter accurately represents intracellular bacterial numbers, RAW264.7 macrophages were infected with Y. pestis CO92 pCD1^(-) Lux_PtolC at the indicated MOIs (n = 12), and extracellular bacteria were killed with gentamicin. (C) Bioluminescence (in relative light units [RLU]) of intracellular bacteria was determined at 1, 4, 8, and 18 h postinfection. (D) At 18 h, cells from each MOI (n = 3) were lysed, and bacterial numbers (CFU) were determined and compared to 18-h bioluminescence (in RLU). (E) To demonstrate that RNAi targeting specific genes could impact the intracellular survival of Y. pestis, RAW264.7 macrophages were transfected with siRNAs targeting Rab2A or COPβ1. Forty-eight hours posttransfection, macrophages were infected with Y. pestis CO92 pCD1^(-) Lux_PtolC (MOI of 10). Extracellular bacteria were killed with gentamicin, and intracellular bacterial bioluminescence was monitored over time. Data are represented as percent RLU of scrambled (Scr) siRNA control. (F) To demonstrate the robustness of the assay, RAW264.7 macrophages (n = 48) were reverse transfected with either scrambled siRNA (negative control) or siRNA targeting Copβ1 (positive control). Forty-eight hours posttransfection, macrophages were infected with Y. pestis CO92 pCD1^(-) Lux_PtolC (MOI of 10). Extracellular bacteria were killed with gentamicin, and intracellular bacterial bioluminescence was determined at 2 and 10 h postinfection. The Z’ factors from four independent experiments are shown (the bars are means). (G) Overview of optimized high-throughput assay for RNAi screening.

FIG 2

Identification of host factors required for intracellular survival of Y. pestis. RAW264.7 macrophages were reverse transfected with siRNAs for 48 h. (A) Transfected cells were infected with Y. pestis CO92 pCD1^(-) Lux_PtolC (MOI of 10), and intracellular bacterial bioluminescence (in RLU) was determined at 2 and 10 h postinfection. RLU values were normalized to the values for the controls and ranked from lowest to highest. Normalized scores of ≤0.4 are indicated by light blue shading, and normalized scores of ≥1.4 are indicated by yellow shading. (Inset) Average Z factor (Z′) ± standard deviation (SD) for all 205 screened plates. (B and C) For secondary validation, cells transfected with siRNA “A” (B) or siRNA “B” (C) were infected with Y. pestis CO92 pCD1^(-) Lux_PtolC or KIMD19 pCD1⁽⁺⁾ Lux_PtolC (MOI of 10), and intracellular bacterial bioluminescence (in RLU) was determined at 10 h postinfection. RLU values for each strain were normalized to the values for the controls and compared. Normalized scores of ≤0.6 are indicated by blue shading. (D) Cytoscape-generated layout for Gene Ontology (GO) term node clusters, with significant genes per cluster highlighted in red. Clusters are color coded by parent ontology, and subgroup ontology is labeled in black. Lines represent interconnections between detailed terms. Node size denotes significance. (E) Pie chart representing the percent parent ontology represented as a whole within validated genes. pV, P value.

FIG 3

Rab4a, Rab11b, and Myo5b are required for intracellular survival of Y. pestis. RAW 264.7 macrophages were transfected with siRNAs targeting Rab4a, Rab11b, or Myo5B. (A and B) Forty-eight hours after transfection, RNA samples were harvested for qRT-PCR (n = 9) (represented as relative expression of scrambled-siRNA-treated cells) (A) or cell viability was determined (n = 5) (B). (C and D) To determine the impact of RNAi on Y. pestis survival, transfected RAW264.7 macrophages (n = 6) were infected with Y. pestis CO92 pCD1^(-) Lux_PtolC (MOI of 10), and intracellular bacterial numbers were determined by bioluminescence (in RLU) at 2 h (C) or 10 h (D) postinfection and compared to the values for scrambled (Scr) controls. (E) At 10 h postinfection, a subset of samples (n = 3) were harvested for conventional bacterial enumeration. (F) Percent of intracellular bioluminescence at 10 h postinfection compared to 2 h postinfection. Values that are significantly different by one-way ANOVA with Tukey’s posthoc test are indicated by asterisks as follows: **, P ≤ 0.01; ***, P ≤ 0.001. Values that are not significantly different (ns) are indicated.

FIG 4

Rab4a is essential for Y. pestis to avoid phagosome acidification and LAMP-1 acquisition. (A) Representative confocal microscopy images of RAW264.7 macrophages transfected with the indicated siRNAs that were treated with Lysotracker and infected with Y. pestis CO92 pCD1^(-) pGEN222, which expresses EGFP (MOI of 7.5). The different colors indicate the following: Lysotracker (red), Y. pestis (green), and colocalization (yellow). Bars = 10 µm. (B and C) Yersinia-containing vacuole (YCV) colocalization with Lysotracker was calculated using IMARIS at 20 min (B) and 80 min (C) postinfection. (D) Representative confocal microscopy images of RAW264.7 macrophages transfected with scrambled or Rab4a siRNAs that were infected with Y. pestis CO92 pCD1^(-) pGEN222 (MOI of 3) and stained with anti-LAMP-1 antibody. LAMP-1 is shown in red, and Y. pestis is shown in green. Yellow arrows indicate locations of bacteria that do not colocalize with LAMP-1 and white arrowheads indicate bacteria that colocalize with LAMP-1 based on Imaris COLOC function. Bars = 5 µm. (E and F) YCV colocalization with LAMP-1 was calculated using IMARIS at 20 min (E) and 80 min (F) postinfection. One-way ANOVA with Tukey’s posthoc test was performed, and the results are indicated as follows: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. The treatments are indicated as follows: Scr, scrambled siRNA; Killed, untransfected macrophages infected with PFA-killed Y. pestis CO92 pCD1^(-) pGEN222; Rab4a, Rab4a siRNA; Rab11b, Rab11b siRNA; Myo5b, Myo5b siRNA.

FIG 5

Y. pestis recruits Rab4a and Rab11b to the YCV. (A and B) Representative confocal microscopy images of primary peritoneal macrophages infected with Y. pestis CO92 pCD1^(-) pGEN::mCherry (Live) (MOI of 3), PFA-killed Y. pestis CO92 pCD1^(-) pGen::mCherry (Killed) (MOI of 3), or E. coli K-12 pGEN::mCherry (E. coli) (MOI of 20) and labeled with anti-Rab4a (A) or anti-Rab11b (B) antibodies. Merged (bacteria [red] and Rab protein [green]) and YCV-Rab colocalization fields generated by Imaris (COLOC) are shown. Bars = 5 µm. (C) Frequency of colocalization of bacterium-containing vacuoles with endogenous Rab4a in peritoneal macrophages. (D) Frequency of colocalization of bacterium-containing vacuoles with Rab4a-EGFP in RAW264.7 macrophages transfected with pEGFP-Rab4a. Yp, Y. pestis; Ec, E. coli. (E) Frequency of colocalization of bacterium-containing vacuoles with endogenous Rab11b in peritoneal macrophages. (F) Frequency of colocalization of bacterium-containing vacuoles with Rab11b-EGFP in RAW264.7 macrophages transfected with pEGFP-Rab11b. (G) Frequency of YCV colocalization with Rab4a-EGFP during 10 h of Y. pestis CO92 pCD1^(-) pGEN::mCherry infection of RAW264.7 macrophages transfected with pEGFP-Rab4a. (H) Frequency of YCV colocalization with Rab11b-EGFP during 10 h of Y. pestis CO92 pCD1^(-) pGEN::mCherry infection of RAW264.7 macrophages transfected with pEGFP-Rab11b. (I and J) Representative images of RAW264.7 macrophages transiently transfected with pEGFP-Rab4a (I) or pEGFP-Rab11b (J) and coinfected with Y. pestis CO92 pCD1^(-) (blue) (MOI of 3) or E. coli K-12 pGEN::mCherry (red) (MOI of 20). Bars = 10 µm. (K and L) Frequency of colocalization of Y. pestis- or E. coli-containing vacuoles in coinfected cells withEGFP-Rab4a (K) or EGFP-Rab11b (L). One-way ANOVA with Tukey’s posthoc test was performed, and the results are indicated as follows: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 6

Y. pestis infection disrupts host cell recycling. (A) Representative images of uninfected RAW264.7 macrophages showing total TfR (Unwashed) and intracellular TfR remaining after washing with high-salt, low-pH buffer to remove antibody labeling of extracellular receptors (Washed). (B) Representative images of uninfected RAW264.7 macrophages infected with Y. pestis CO92 pCD1^(-) (Live Yp) (MOI of 20), PFA-killed Y. pestis CO92 pCD1^(-) (Killed Yp) (MOI of 20), or E. coli K-12 (MOI of 100) for 24 h and then washed with high-salt, low-pH buffer to remove antibody labeling of extracellular receptors. The nuclei are stained with DAPI (blue) and TfR (green). Bars = 10 µm. (C to H) Mean field intensity of TfR signal per cell was calculated by confocal microscopy at 2 and 24 h postinfection with PFA-killed Y. pestis CO92 pCD1^(-) (Killed Yp) (C and D), live Y. pestis CO92 pCD1^(-) (Live Yp) (E and F), or E. coli K-12 (G and H). UI, uninfected. (I and J) Mean field intensity of TfR signal per cell from hMDMs infected with live Y. pestis CO92 pCD1^(-) (Yp) (MOI of 10), PFA-killed Y. pestis CO92 pCD1^(-) (Killed) (MOI of 10), E. coli K-12 (Ec) (MOI of 100), or S. enterica Typhimurium (Sal) (MOI of 100) for 2 h (I) or 24 h (J). Data from one experiment representative of three independent experiments are shown. Each data point represents the mean field intensity (TfR) per cell from an individual field (n = 25; ~100 cells per field). The bars represent the means. One-way ANOVA with Dunnett’s posthoc test was performed (values compared to uninfected values), and the results are shown as follows: ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 7

Overexpression of Rab11b restores recycling and inhibits intracellular growth of Y. pestis. (A to D) RAW264.7 macrophages were transiently transfected with pEGFP-Rab11b prior to infection with Y. pestis CO92 pCD1^(-) pGEN::mCherry (MOI of 10), and TfR was differentially labeled and imaged by confocal microscopy. Individual Rab11b-overexpressing transfected (OE) and untransfected (Un) cells were manually isolated using IMARIS to maintain host cell boundaries and analyzed for intracellular TfR and bacteria (n = 50 cells each). (A) Intracellular TfR intensity per cell. (B) Number of TfR-positive endosomes per cell. (C) Intracellular bacterial numbers as a function of mCherry signal area per cell. (D) Representative image of infected cells overexpressing Rab11b at 24 h postinfection. (E) Intracellular bacterial numbers as a function of mCherry signal area per cell for cells overexpressing Rab4a (OE) compared to untransfected cells (Un). (F) Representative image of infected cells from Rab4a overexpression studies at 24 h postinfection. Data from one experiment representative of three independent experiments are shown. Paired Student’s t test was used to compare samples from the same time point and ANOVA with Tukey’s posthoc test was used for comparisons different time points (in panels C and E only), and results are indicated as follows: ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001. Red bars show the mean values. For microscopy images, Y. pestis (red), Rab protein (green), bacteria in untransfected cells (white arrowheads), and bacteria in Rab-overexpressing cells (yellow arrowheads) are indicated. Cell borders are shown outlined in white.

FIG 8

Model of the biogenesis of the Yersinia-containing vacuole (YCV). Y. pestis engages the host endosome recycling pathway by recruiting Rab GTPases to the YCV in order to generate a protective replicative niche in a two-step process. First, Rab1b and Rab4a are recruited to the YCV, which is required for the bacterium to inhibit phagosome acidification and fusion with the lysosome. While these two Rab proteins are eventually lost from the YCV, Rab11b is retained on the YCV over the entire course of infection. Retention of Rab11b leads to a global inhibition of host recycling, which is required for Y. pestis to replicate in macrophages. Rab4, Rab11, and Rab1b proteins are shown in the figure as gray circles labeled 4, 11, and 1B, respectively.