We have recently demonstrated that Salmonella grown with physiological levels of Ca++ are significantly enhanced in their ability to penetrate HeLa cells in vitro. This stimulation in HeLa cell penetration was dose dependent, required bacterial metabolism and RNA synthesis, could not be shown with other divalent cations and was a stable property of treated cells. In this proposal, we will investigate the mechanism by whch Ca++ mediates Salmonella invasion enhancement. Ca++-mediated alterations is the expression of Salmonella inner and outer membrane polypeptides and lipopolysaccharide determinants will be determined after isolation and analyses by biochemical and gel electrophoretic techniques. To identify Ca++-responsive genes and determine their role in Salmonella invasion enhancement, we will construct operon gene fusions using lambda placMU vectors. These fusion strains will be used to evaluate the intracellular expression of Ca++-responsive genes. Ca++-mediated enhanced expression of other Salmonella virulence products (ie. cytotoxin and enterotoxin) will also be examined. To investigate the in vivo significance of Ca++-enhanced Salmonella invasion, we will compare Ca++-enhanced and control Salmonella: to elicit fluid accumulation, produce and inflammatory reaction, cause tissue destruction in rabbit ligated loops, and to disseminate from the intestine into the blood or other organs. We will also determine the response of different host defense mechanisms to Ca++- enhanced Salmonella. The ability of epithelial cells to resist penetration after IFN treatment will be examined. Ca++- mediated enhanced uptake or survival in macrophages will also be considered. We will also look for new antigenic determinants and the ability of nonlymphoid cells to produce IFN after invasion by CA++-enhanced Salmonella. Findings from these studies will significantly contribute toward furthering our understanding of the mechanism of bacterial invasion in epithelial cells, the role of Ca++ in facultative intracellular bacterial disease and enrich our understanding of bacterial host cell physiology.
