Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis

The study of intracellular bacterial pathogens in cell culture hinges on inhibiting extracellular growth of the bacteria in cell culture media. Aminoglycosides, like gentamicin, were originally thought to poorly penetrate eukaryotic cells, and thus, while inhibiting extracellular bacteria, these antibiotics had limited effect on inhibiting the growth of intracellular bacteria. This property led to the development of the antibiotic protection assay to study intracellular pathogens in vitro. More recent studies have demonstrated that aminoglycosides slowly penetrate eukaryotic cells and can even reach intracellular concentrations that inhibit intracellular bacteria. Therefore, important considerations, such as antibiotic concentration, incubation time, and cell type need to be made when designing the antibiotic protection assay to avoid potential false positive/negative observations. Yersinia pestis, which causes the human disease known as the plague, is a facultative intracellular pathogen that can infect and replicate in macrophages. Y. pestis is sensitive to gentamicin and this antibiotic is often employed in the antibiotic protection assay to study the Y. pestis intracellular life cycle. However, a large variety of gentamicin concentrations and incubation periods have been reported in the Y. pestis literature without a clear characterization of the potential influences that variations in the gentamicin protection assay could have on intracellular growth of this pathogen. This raised concerns that variations in the gentamicin protection assay could influence phenotypes and reproducibility of data. To provide a better understanding of the potential consequences that variations in the gentamicin protection assay could have on Y. pestis, we systematically examined the impact of multiple variables of the gentamicin protection assay on Y. pestis intracellular survival in macrophages. We found that prolonged incubation periods with low concentrations of gentamicin, or short incubation periods with higher concentrations of the antibiotic, have a dramatic impact on intracellular growth. Furthermore, the degree of sensitivity of intracellular Y. pestis to gentamicin was also cell type dependent. These data highlight the importance to empirically establish cell type specific gentamicin protection assays to avoid potential artificial data in Y. pestis intracellular studies

<i>Yersinia pestis</i> Requires Host Rab1b for Survival in Macrophages

<div><p><i>Yersinia pestis</i> is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages <i>Y</i>. <i>pestis</i> actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by <i>Y</i>. <i>pestis</i> 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 <i>Y</i>. <i>pestis</i> to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the <i>Yersinia</i> containing vacuole (YCV) and that <i>Y</i>. <i>pestis</i> 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 <i>Legionella pneumophila</i> 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.</p></div

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

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

Knockdown of Rab1b increases <i>L</i>. <i>pneumophila</i> LCV acidification.

<p>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 <i>L</i>. <i>pneumophila</i> pMIP-GFP (MOI 10). Coverslips were fixed and colocalization of Lysotracker was determined by confocal microscopy. (A) Representative images showing colocalization of Lysotracker with <i>L</i>. <i>pneumophila</i>. 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.</p

Rab1b knockdown inhibits the survival of <i>Y</i>. <i>pestis</i> within macrophages.

<p>RAW264.7 macrophages were reverse transfected with Rab1a, Rab1b, scrambled (Scr), or Copβ1 siRNA. 48 h after transfection cells were infected with <i>Y</i>. <i>pestis</i> (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 <i>Y</i>. <i>pestis</i> CO92 pCD1^(-) Lux_PtolC. (C) Conventional enumeration of intracellular bacteria from macrophages infected for 10 h with <i>Y</i>. <i>pestis</i> CO92 pCD1^(-) Lux_PtolC. (D) Bioluminescence of intracellular bacteria from macrophages infected for 2 h with <i>Y</i>. <i>pestis</i> CO92 pCD1^(-) Lux_PtolC. (E) Bioluminescence of intracellular bacteria from macrophages infected for 10 h with <i>Y</i>. <i>pestis</i> KIM D-19 Lux_PtolC. (F) Conventional enumeration of intracellular bacteria from macrophages infected for 10 h with <i>Y</i>. <i>pestis</i> KIM D-19 Lux_PtolC. (G) Bioluminescence of intracellular bacteria from macrophages infected for 2 h with <i>Y</i>. <i>pestis</i> 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.</p

Rab1b is recruited to the YCV.

<p>RAW264.7 macrophages were transiently transfected with pEGFP-Rab1B(CA). 24 h after transfection cells were infected with either live or paraformaldehyde killed <i>Y</i>. <i>pestis</i> pMCherry (MOI 7.5) or <i>E</i>. <i>coli</i> 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.</p

Rab1b knockdown does not impact <i>Y</i>. <i>pestis</i> invasion of macrophages.

<p>RAW264.7 macrophages were reverse transfected with Rab1b, scrambled (Scr), or Copβ1 siRNA. 48 h after transfection cells were infected with <i>Y</i>. <i>pestis</i> CO92 pCD1^(-)pGEN-<i>P</i>_<i>EM7</i>::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-<i>Y</i>. <i>pestis</i> antibody. (A) Representative image showing differential staining of intracellular (red) and extracellular (green or yellow) bacteria. Scale bar is 5μm. Asterisk denotes intracellular <i>Y</i>. <i>pestis</i>. (B and C) Percentage of intracellular bacteria calculated at 20 and 80 min post-infection, respectively. ** = p<0.01, ns = not significant.</p

Rab1b knockdown alters YCV acidification.

<p>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 <i>Y</i>. <i>pestis</i> CO92 pCD1^(-) pGEN222 expressing EGFP (MOI 7.5). Colocalization of Lysotracker Red DND-99 and <i>Y</i>. <i>pestis</i> CO92 pCD1^(-) pGEN222 was determined by confocal microscopy. (A) Representative images showing colocalization of Lysotracker Red DND-99 and <i>Y</i>. <i>pestis</i>. 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.</p

Rab1b knockdown does not affect YCV association with LC3.

<p>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 <i>Y</i>. <i>pestis</i> CO92 pCD1^(-) pGEN-<i>P</i>_<i>EM7</i>::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.</p

Type 3 secretion system induced leukotriene B4 synthesis by leukocytes is actively inhibited by Yersinia pestis to evade early immune recognition.

Subverting the host immune response to inhibit inflammation is a key virulence strategy of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the Y. pestis mechanisms to manipulate the inflammatory cascade is necessary to understand this pathogen's virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of the inflammatory lipid mediator response during plague. Here we used the murine model to define the kinetics of the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and immune cell activator, within the lungs during pneumonic plague. Furthermore, we demonstrated that exogenous administration of LTB4 prior to infection limited bacterial proliferation, suggesting that the absence of LTB4 synthesis during plague contributes to Y. pestis immune evasion. Using primary leukocytes from mice and humans further revealed that Y. pestis actively inhibits the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the rapid synthesis of LTB4. However, several Yop effectors secreted through the T3SS effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of the inflammatory lipid LTB4 contributing to the delay in the inflammatory cascade required for rapid recruitment of leukocytes to sites of infection