| The coordinated function of polymorphonuclear leukocytes (PMNs, or neutrophils) and macrophages of the innate immune system recognize and eliminate most invading bacteria. The pathogen Yersinia pestis, causative agent of plague, resists destruction by these innate phagocytes. The specific molecular mechanisms used by Y. pestis to survive following phagocytosis are incompletely defined. Herein, we demonstrate that Y. pestis uses both active and passive subversion strategies to promote survival within the mammalian host. We inactivated each of the known two-component gene regulatory systems (TCSs) and assessed susceptibility of these mutant strains to human PMN granule extracts Y. pestis strains deficient for PhoPQ, KdpED, CheY, CvgSY, and CpxRA TCSs were selected for further analysis and all 5 strains were susceptible to killing by PMNs. However, only DeltaphoPQ demonstrated global sensitivity to a panel of 7 individual neutrophil antimicrobial peptides and serine proteases and for intracellular survival in PMNs. Iterative analysis with Y. pestis strains lacking the PhoP-regulated genes ugd and pmrK indicated that the mechanism most likely responsible for increased resistance to killing is modification of lipid A. Furthermore, Y. pestis is a recent clone of Y. pseudotuberculosis with significant gene loss, including the principal protein component of the flagellar filament and known pathogen-associated molecular pattern (PAMP) in flagella. We tested whether loss of flagella filament expression contributes to immune evasion by limiting bacterial detection by phagocytes. In support of this hypothesis, we confirmed that the flagellar master regulator, flhD , contained a frameshift mutation in all sequenced Y. pestis strains. A recombinant strain expressing a flagellin-beta-lactamase fusion was incubated with human neutrophils or murine macrophages containing a beta-lactamase cleavable CCF2-AM-fluorescent substrate so that flagellin location could be observed by fluorescent microscopy. The Y. pestis type-three secretion system (TTSS) was required for flagellin translocation into the cytosol of both host cell types. Translocation of flagellin induced increases in macrophage IL-6, MIP-2, IL-1beta and macrophage cell death Thus, this research futhers our understanding of immune cell subversion mechanisms used by Y. pestis including resisting microbicidal granule contents and by inactivating flagellin expression. |
