| Measures aimed at preventing the occurrence and reducing the severity of infectious diseases require an understanding of the means by which pathogenic microorganisms gain access to, survive in, and are transmitted to susceptible hosts. Current approaches are designed to answer fundamental questions regarding the interplay between bacterial pathogen and host organism. Common to these approaches is the identification and functional characterization of bacterial genes that allow the pathogen to cause disease. One of these approaches, termed i&barbelow;n v&barbelow;ivo e&barbelow;xpression t&barbelow;echnology (IVET) provided the foundation for the results described in this thesis by identifying >200 genes in the human pathogen Salmonella typhimurium whose expression are upregulated during infection [in vivo induced (ivi) genes]. This collection of genes was used to identify novel segments of the Salmonella chromosome that are specific to the salmonellae, contribute to pathogenesis, and whose presence or absence in different strains that infect a variety of hosts may provide clues to the evolution of this pathogen. At the boundary of two contiguous Salmonella-specific regions is a small, untranslated RNA molecule, ssrA, which was found to regulate the expression of several ivi genes and is required for pathogenesis. Additionally, since S. typhimurium DNA adenine methylase (Dam) mutants have been shown to be completely attenuated and can serve as effective live attenuated vaccines, Dam's role in the virulence of two different human pathogens, Vibrio cholerae and Yersinia pseudotuberculosis, was determined. Unlike dam mutations in S. typhimurium, which are viable, dam was found to be essential for viability in V. cholerae and Y. pseudotuberculosis. Overproduction of Dam was not lethal, but significantly attenuated the virulence of these two pathogens and, in Yersinia, led to a fully protective immune response. In addition, key Yersinia virulence determinants are dysregulated in response to Dam overproduction, supporting the hypothesis that inappropriate expression of Dam-controlled genes leads to the inability to cause disease as well as the ability to induce protective immunity in the host. These results indicate that vaccine methodology based on Dam mutations may offer a new route for the development of therapeutics against a wide range of human and animal pathogens. |
