Individuals with AIDS and other compromised states experience increased systemic disease caused by Salmonella spp. other than S. typhi. The non-typhoid serovars of salmonellae that cause systemic disease (S. typhimurium, S. dublin, S. enteritidis) possess related virulence plasmids. Results from the previous funding period showed that the five spv genes of the S. typhimurium virulence plasmid cause systemic disease in a mouse model by increasing the growth rate of salmonellae within host cells. However, the molecular and cellular mechanisms of Spv-mediated virulence of salmonellae remain unsolved, and no existing in vitro model reproduces Spv-mediated increased growth rate. The long term goals of the continuation of this project are to elucidate the Spv virulence function and to determine the mechanisms by which the host suppresses growth of intracellular bacteria.
The specific aims are to: 1. Identify the mouse cell(s) and their functions which affect virulence plasmid-mediated intracellular proliferation. They will identify the primary host cells in which the Spv function is manifested and secondary cells which suppress Spv-mediated growth by using immunochemical staining of infected mouse tissues; genetic, immunologic, and pharmacologic alteration of mice during infection with Spv+ and Spv-salmonellae. 2. Identify non-spv salmonella genes and their functions that are involved with the Spv phenotype. They will identify proteins differentially expressed by Spv+ and Spv- salmonellae, and Spv-regulated genes will be isolated using gene fusions. They will isolate salmonella mutants defective for intracellular replication in mice. They will combine these mutations with Spv- salmonellae and look for additive effects on virulence. 3. Develop an in vitro model for Spv-mediated intracellular proliferation and utilize the model to elucidate the spv virulence function at the cellular and molecular levels. They will infect animal cell cultures based on the results of aim 1. Once Spv-dependent intracellular growth is reproduced in vitro, we will examine the ultrastructural location of physiological environment of the intracellular bacteria, as well as the host cell response to intracellular infection. They will attempt to reproduce Spv-mediated growth in a cell-free medium and ultimately elucidate the bacterial physiology of virulence plasmid-mediated growth. These studies will elucidate the pathogenic mechanism of the spv genes, identify associated chromosomal virulence genes, and identify host defenses against intracellular bacterial growth. The dissection of the roles of the host and pathogen in intracellular growth will lead to new information about the mechanism of systemic disease caused by salmonellae, as well as other intracellular bacterial pathogens.
