We will study the role of the PhoP regulon in Salmonella typhimurium virulence and murine typhoid fever. Our goal is to define the virulence genes and protein products regulated by PhoP/PhoQ. This will include the isolation and analysis of strains containing TnphoA insertions that are both positively (phoP activated genes or pags) and negatively (phoP repressed genes or prgs) regulated by PhoP. We will perform TnphoA mutagenesis on a strain of Salmonella that constitutively expresses pags and identify pags by screening for loss of fusion protein activity upon introduction of a phoP mutation. We will perform TnphoA mutagenesis on a strain deleted in phoP and identify prgs by screening for loss of fusion protein activity upon introduction of a phoP constitutive mutation. Salmonella strains with gene fusions regulated by phoP will be studied for mouse virulence, macrophage survival, and NP-1 defensin sensitivity. We have already begun to analyze two pag loci, termed pagC and pagD and propose experiments to further define their role in virulence. The DNA sequence, protein products, transcripts, and promotor elements of pag and prg loci will be determined to better understand the molecular pathogenesis of Salmonella infections. phoP constitutive mutations confer reduced virulence and marked immunogenicity on S. typhimurium and specific DNA mutations that confer this phenotype will also be defined. The pagC gene product is similar to the ail invasion locus of Yersinia enterocolitica and the lom encoded bacteriophage lambda membrane protein. Experiments are planned to study the role of PagC and Lom in macrophage survival and invasion. Whenever possible we will correlate new observations about pathogenesis to the human agent of typhoid fever, Salmonella typhi. We plan to apply the knowledge we obtain the development and improvement of live vaccines for typhoid fever.
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