Identification and characterization of genes involved in survival and replication of Yersinia pestis and Yersinia pseudotuberculosis in macrophages

Yersinia pestis and Yersinia pseudotuberculosis are closely related gram-negative bacteria that cause vastly different human diseases. Even though they use distinct routes of entry during infection, both pathogens share a common tropism for lymphoid tissue. Previous studies suggested that Y. pestis and Y. pseudotuberculosis are internalized by macrophages at early times during the infection process, but the bacteria are not degraded. In vitro experiments using cultured macrophages provided evidence that both pathogens are able to survive and replicate in macrophages. In addition, the phoP gene was shown to be required for survival of Y. pestis in macrophages and for virulence in mice. phoP encodes the response regulator of the PhoP/PhoQ two-component system, which in Salmonella is activated under low magnesium conditions and in macrophages.; I constructed Y. pestis and Y. pseudotuberculosis phoP mutants and tested them for their ability to grow in low magnesium medium and in macrophages. The Y. pseudotuberculosis phoP mutant was also tested for virulence in mice. In addition, experiments were performed to better characterize the intracellular compartment in which Y. pestis resides within the macrophage. To identify genes that were regulated by PhoP, a Tn5-lacZ screen was performed in Y. pestis.; My results show that phoP is important for proliferation of Y. pseudotuberclosis in macrophages and virulence in mice. PhoP is essential for survival of Y. pestis in macrophages due to the lack of O-antigen in this bacterium. Data presented here suggest that Y. pestis resides in a phagosome that takes on many characteristics of a phagolysosome. PhoP-regulated genes important for survival in this compartment fall into two groups: genes needed for survival under low magnesium conditions and genes required for modification of the outer membrane.; We have demonstrated that PhoP is an important virulence factor in Y. pseudotuberculosis and is required for proliferation of Y. pestis and Y. pseudotuberculosis in macrophage and in low magnesium medium. To survive in the presumably low magnesium environment of the phagosome, PhoP-regulated genes are induced within the macrophage to allow Y. pestis and Y. pseudotuberculosis to adapt to this new environment by modifying its outer membrane and expressing high affinity magnesium transporters.