Characterization of Phagosome Trafficking and Identification of PhoP-Regulated Genes Important for Survival ofYersinia pestisin Macrophages

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Abstract
The transcriptional activator PhoP is important for survival ofYersinia pestisin macrophage phagosomes. However, the phagosomes inhabited byY. pestishave 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 containingphoP+orphoPmutantY. pestisstrains or latex beads in J774A.1 macrophages. Phagosomes containingphoP+orphoPmutantY. pestisacquired lysosomal markers to the same degree that phagosomes containing latex beads acquired these markers after 1.5 h of infection, showing that nascent phagosomes containingY. pestisfuse with lysosomes irrespective of thephoPgenotype. Similar results were obtained when phagosomes containing viable or deadphoP+Y. pestiscells or beads were analyzed at 8 h postinfection, indicating that theY. pestisvacuole does not become secluded from the lysosomal compartment. However, only viablephoP+bacteria induced the formation of spacious phagosomes at 8 h postinfection, suggesting thatY. pestiscan actively direct the expansion of its vacuole. PhoP-regulated genes that are important for survival ofY. pestisin phagosomes were identified by Tn5-lacZmutagenesis and oligonucleotide microarray analysis. Three such genes were identified, and the products of these genes are predicted to promote resistance to antimicrobial peptides (ugdandpmrK) or low-Mg2+conditions (mgtC) found in phagosomes. Viable count assays carried out withY. pestis ugd,mgtC, andugd mgtCmutants revealed that the products ofugdandmgtCfunction independently to promote early survival ofY. pestisin macrophage phagosomes.