Abstract
ABSTRACTYersinia 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.
Highlights
IMPORTANCE Yersinia pestis can infect and survive within macrophages
We infected cells transfected with Rab2 small interfering RNA (siRNA) and Cop1 siRNA with Y. pestis CO92 pCD1(-) LuxPtolC, a bioluminescent bioreporter that can differentiate as little as a twofold difference in intracellular bacteria (Fig. 1C and D; R2 ϭ 0.89) [50], to demonstrate that this bioreporter can be used to kinetically monitor changes in intracellular survival (Fig. 1E)
While it has been known for decades that Y. pestis survives within a vacuolar compartment within macrophages [27, 28], the mechanisms leading to subversion of phagolysosome killing by macrophages have not been defined
Summary
IMPORTANCE Yersinia pestis can infect and survive within macrophages. the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. Y. pestis can be directly inoculated into blood by a flea or from the bite of an infected animal, resulting in primary septicemic plague without colonization of the lymphatic system [1]. The ability of Y. pestis to exist in both the mammalian and flea hosts is a result of acquiring virulence factors required for the mammalian host and transmission factors required for flea colonization [1, 8,9,10] The bacterium regulates these factors to ensure expression of appropriate factors only when required [1, 8,9,10,11,12].
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