application) Salmonella typhimurium is a common cause of food-borne enterocolitis in this country and the developing world. The clinical manifestations of Salmonella typhimurium infection result from inflammatory cells that are recruited to, and accumulate in, the intestinal mucosa. The intestinal inflammatory response is mediated in large part by synthetic upregulation of secreted cytokines and other inflammatory effector molecules. These inflammatory mediators are activated at the transcriptional level by the action of DNA binding transcription factors such as NF-kappaB. Experiments with cultured intestinal epithelial cells have shown that S. typhimurim associated with inflammatory intestinal disease activate NF-kappaB via a calcium dependant activation pathway, while other, non-pathogenic Salmonella strains repress activation of this key regulator. Our overall hypothesis is that Salmonella (and potentially other enteric bacteria) have evolved novel mechanisms to modulate epithelial signaling pathways that may serve to establish either a pathologic or a commensal state. Elucidation of these influences will increase our understanding of the epithelial and bacterial factors involved in human enterocolitis, as well as the microbiology of this class of enteric infections. This application describes a variety of experimental methods to study the host inflammatory response including pathogen induced calcium dependant activation of the NF-kappaB pathway. These methods will be applied to signals elicited by S. typhimurium, a series of Salmonella mutants and variety of other enteric pathogens. Secondly, similar assays will be used to study the mechanism by which some bacteria repress host immune and inflammatory reactions by modulation of the NF-kappaB signaling cascade. These studies will be supported using well characterized murine models of intestinal inflammation to correlate the anti-inflammatory properties of these bacteria in vivo. Finally, a novel electroporation based method to introduce purified proteins into intact model epithelia will be used to dissect the cellular responses to internalization of a variety of bacterial effector proteins.
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