This proposal aims to characterize the effects of the Salmonella typhimurium type III-secreted AvrA effector protein on NF-kB activation and test the general hypothesis that some Salmonella strains have evolved a mechanism to modulate epithelial proinflammatory signaling pathways. S. typhimurium is a common cause of foodborne enterocolitis in this country and the developing world. The inflammation typically associated with S. typhimurium infections results from recruitment of PMNs by the chemotactic cytokine IL-8. IL-8 and other inflammatory mediators can be activated by NF-kB. While it has been demonstrated that some Salmonella strains activate NF-kB, we have identified several nonpathogenic strains which inhibit NF-kB activation. Based on our preliminary data showing that the AvrA effector blocks TNF-induced NF-kB activation, the goals of the work proposed here are to determine if AvrA is responsible for the anti-inflammatory effects of these nonpathogenic Salmonella strains on activation of NF-kB and to define the inhibitory effect of AvrA on NF-kB activation. In cells transfected with AvrA, nuclear translocation of NF-kB will be assessed by immunofluorescence techniques and effects on IkB-a phosphorylation, ubiquitination, and degradation will be determined by Western blot. In addition, AvrA knockout Salmonella strains will be constructed and the ability of these mutants to inhibit NF-kB activation will be tested.
