A transposon site hybridization screen identifies galU and wecBC as important for survival of Yersinia pestis in murine macrophages.

Yersinia pestis is able to survive and replicate within murine macrophages. However, the mechanism by which Y. pestis promotes its intracellular survival is not well understood. To identify genes that are important for Y. pestis survival in macrophages, a library comprised of ∼31,500 Y. pestis KIM6+ transposon insertion mutants (input pool) was subjected to negative selection in primary murine macrophages. Genes underrepresented in the output pool of surviving bacteria were identified by transposon site hybridization to DNA oligonucleotide microarrays. The screen identified several genes known to be important for survival of Y. pestis in macrophages, including phoPQ and members of the PhoPQ regulon (e.g., pmrF). In addition, genes predicated to encode a glucose-1-phosphate uridylyltransferase (galU), a UDP-N-acetylglucosamine 2-epimerase (wecB) and a UDP-N-acetyl-d-mannosamine dehydrogenase (wecC) were identified in the screen. Viable-count assays demonstrated that a KIM6+ galU mutant and a KIM6+ wecBC mutant were defective for survival in murine macrophages. The galU mutant was studied further because of its strong phenotype. The KIM6+ galU mutant exhibited increased susceptibility to the antimicrobial peptides polymyxin B and cathelicidin-related antimicrobial peptide (CRAMP). Polyacrylamide gel electrophoresis demonstrated that the lipooligosaccharide (LOS) of the galU mutant migrated faster than the LOS of the parent KIM6+, suggesting the core was truncated. In addition, the analysis of LOS isolated from the galU mutant by mass spectrometry showed that aminoarabinose modification of lipid A is absent. Therefore, addition of aminoarabinose to lipid A and complete LOS core (galU), as well as enterobacterial common antigen (wecB and wecC), is important for survival of Y. pestis in macrophages.

Characterization of phagosome trafficking and identification of PhoP-regulated genes important for survival of Yersinia pestis in macrophages.

The transcriptional activator PhoP is important for survival of Yersinia pestis in macrophage phagosomes. However, the phagosomes inhabited by Y. pestis have not been well characterized, and the mechanism by which PhoP promotes bacterial survival in these vacuoles is not fully understood. Lysosomal tracers, as well as antibodies to late endosomal or lysosomal proteins, were used in conjunction with confocal or electron microscopy to study the trafficking of phagosomes containing phoP(+) or phoP mutant Y. pestis strains or latex beads in J774A.1 macrophages. Phagosomes containing phoP(+) or phoP mutant Y. pestis acquired lysosomal markers to the same degree that phagosomes containing latex beads acquired these markers after 1.5 h of infection, showing that nascent phagosomes containing Y. pestis fuse with lysosomes irrespective of the phoP genotype. Similar results were obtained when phagosomes containing viable or dead phoP(+) Y. pestis cells or beads were analyzed at 8 h postinfection, indicating that the Y. pestis vacuole does not become secluded from the lysosomal compartment. However, only viable phoP(+) bacteria induced the formation of spacious phagosomes at 8 h postinfection, suggesting that Y. pestis can actively direct the expansion of its vacuole. PhoP-regulated genes that are important for survival of Y. pestis in phagosomes were identified by Tn5-lacZ mutagenesis and oligonucleotide microarray analysis. Three such genes were identified, and the products of these genes are predicted to promote resistance to antimicrobial peptides (ugd and pmrK) or low-Mg(2+) conditions (mgtC) found in phagosomes. Viable count assays carried out with Y. pestis ugd, mgtC, and ugd mgtC mutants revealed that the products of ugd and mgtC function independently to promote early survival of Y. pestis in macrophage phagosomes.

Replication of Yersinia pestis in interferon gamma-activated macrophages requires ripA, a gene encoded in the pigmentation locus.

Yersinia pestis is a facultative intracellular bacterial pathogen that can replicate in macrophages. Little is known about the mechanism by which Y. pestis replicates in macrophages, and macrophage defense mechanisms important for limiting intracellular survival of Y. pestis have not been characterized. In this work, we investigated the ability of Y. pestis to replicate in primary murine macrophages that were activated with IFN-gamma. Y. pestis was able to replicate in macrophages that were activated with IFN-gamma after infection (postactivated). A region of chromosomal DNA known as the pigmentation (pgm) locus was required for replication in postactivated macrophages, and this replication was associated with reduced nitric oxide (NO) levels but not with reduced inducible NO synthase (iNOS) expression. Y. pestis delta pgm replicated in iNOS-/- macrophages that were postactivated with IFN-gamma, suggesting that killing of delta pgm Y. pestis is NO-dependent. A specific genetic locus within pgm, which shares similarity to a pathogenicity island in Salmonella, was shown to be required for replication of Y. pestis and restriction of NO levels in postactivated macrophages. These data demonstrate that intracellular Y. pestis can evade killing by macrophages that are exposed to IFN-gamma and identify a potential virulence gene encoded in the pgm locus that is required for this activity.

The response regulator PhoP of Yersinia pseudotuberculosis is important for replication in macrophages and for virulence.

Yersinia pestis and Yersinia pseudotuberculosis are closely related facultative intracellular pathogens. The response regulator PhoP was previously shown to be important for Y. pestis survival in macrophages and for virulence in a murine bubonic plague infection assay. Here the importance of PhoP for Y. pseudotuberculosis pathogenesis was investigated. Y. pseudotuberculosis phoP mutants were unable to replicate in low-Mg(2+) medium or in macrophages. phoP(+) Y. pseudotuberculosis strains initiated replication in macrophages after a lag period of approximately 5 h, as shown by fluorescence microscopy and viable count assays. Y. pseudotuberculosis phoP mutants died at a low rate in macrophages; there was no decrease in viability over the first 5 h of infection, and there was a 10-fold decrease in viability between 5 and 24 h of infection. Trafficking of phagosomes containing phoP(+) or phoP mutant Y. pseudotuberculosis was studied by using immunofluorescence microscopy and cathepsin D as a marker for lysosomes. Phagosomes containing phoP mutant Y. pseudotuberculosis acquired cathepsin D at a higher rate than phagosomes containing phoP(+) bacteria. However, the increased rate of marker acquisition for phagosomes containing mutant bacteria was only evident approximately 5 h after infection, suggesting that phoP mutants are able to retard phagosome maturation during the lag phase of intracellular growth. The results obtained with a Y. pestis phoP mutant were similar to those described above, except that the rates of intracellular killing and trafficking to cathepsin D-positive vacuoles were significantly higher. A Y. pseudotuberculosis phoP mutant was 100-fold less virulent than the wild-type strain in a murine intestinal infection model, suggesting that survival and replication in macrophages are important for Y. pseudotuberculosis pathogenesis.