A number of mutations in Salmonella typhimurium have been described that reduce the virulence of the organism in a murine typhoid model. Many of these mutations also affect survival of S. typhimurium in mouse macrophages. Among the mouse-attenuated S. typhimurium strains that fail to persist in macrophages are strains deleted for flgA, part of the flgB operon, and the mviS locus, i.e. delta flg25. Since plasmids containing the mviS region of DNA from S. typhimurium are able to restore virulence without complementing for flagella synthesis, a wild-type mviS locus appears to be necessary for the full expression of S. typhimurium pathogenicity in the mouse model. The LONG TERM GOALS of this project are to characterize the mviS locus at the molecular level and to begin to evaluate the role of mviS in the virulence of S. typhimurium.
The SPECIFIC AIMS designed to achieve these objectives are: 1) to continue the molecular analysis of the mviS gene by more precisely defining the mviS region, probing other species of Salmonella for an mviS analog, and assessing whether the cloned mviS gene from typically mouse-avirulent Salmonella species enhances the virulence of mviS S. typhimurium; 2) to construct isogenic strains of S. typhimurium that differ only at the mviS locus and to compare the virulence of the flg+mviS+ and flg+mviS- strains for C57BL/6J mice and the survival of each member of the isogenic pair: i) in macrophages from innately salmonella-resistant and salmonella-susceptible macrophages, ii) at low pH, and iii) in the presence of the defensin-like compound protamine; 3) to determine whether mviS functions to regulate expression of other S. typhimurium genes and characterize any mviS-regulated gene(s); 4) to assess the expression and regulation of mviS by generating a fusion of the mviS promoter to the reporter gene beta-galactosidase and monitoring the level of beta-galactosidase: i) under a variety of in vitro growth conditions, ii) inside infected epithelial cells and macrophages, and iii) in tissue homogenates from infected mice. Lastly, the cell type(s) in which the mviS-fusion product is expressed will be determined by histochemical analysis of tissue from infected mice.
