PhoP, A Novel Global Regulator, Directly Activates A Global Regulon Contributing To Intracellular Growth Of Yersinia Pestis

Plague, caused by Yersinia pestis, has ravaged human populations for many centuries and claimed hundred millions of lives in history. The growth and proliferation within the macrophage cell after early-stage entry into host is considered as a key step for further infection and pathogenesis. The two-component system PhoP-PhoQ can govern bacteria virulence, help bacteria to adapt to Mg²⁺ -limiting environments, and regulate numerous cellular activities in several Gram-negative bacteria. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. Although PhoP was previously shown to be important for Y. pestis survival within macrophage and under macrophage-induced stresses, little was known about PhoP-dependent molecular mechanisms by which Y. pestis survives the antibacterial strategies employed by host macrophages.In this study,the phoP gene in Y. pestis was replaced with kanamycin resistance cassette using the one-step disruption protocol. The transcription profile of the wild-type Y. pestis was then compared with that of the phoP mutant under Mg²⁺ -limiting conditions. It was revealed that PhoP-PhoQ governed a wide set of cellular pathways in Y. pestis. Some of these genes are directly controlled by PhoP, others are indirectly. Molecular biochemistry assays, including electrophoretic mobility shift assays (EMSA), DNasel footprinting and primer extension assays, were employed to identify which ones are directly controlled.Firstly, we identified 46 genes directly controlled by PhoP based on EMSA. These genes were either repressed or activated by PhoP. The functional classification of these differentially regulated genes gives an overall picture of the global gene transcription pattern of Y. pestis affected by the PhoP responsive regulator under the Mg²⁺ -limiting conditions. PhoP positively controlled the mgtCB expression in Y. pestis to regulate Mg²⁺ transport systems. PhoP controlled the genes (sodC, sodB, sod A, katA, katY, ahpC and gsi) involved in oxidative stress, the genes of chaperones and ATP-dependent proteases, other stress genes to contribute to the protection against stresses. The glgPACXB operon was induced by PhoP in response to hyperosmotic stress. The phoP gene was also required for aminoarabinose modification of lipid A in Y. pestis. The enzymes encoded by the pmrHFIJKLM operon and the ugd/pmrE gene are responsible for addition of aminoarabinose to lipid A. PhoP could involve in the transcriptional regulation of those genes responsible for energy metabolism, central intermediary metabolism, degradation of small molecules and macromolecules, and biosynthesis of amino acid, purines, pyrimidines, nucleosides and nucleotides, cofactors, prosthetic...