Pathogenic Salmonella strains are an important cause of infectious disease throughout the world. The bacteria possess an invasion-associated type III secretion system, encoded on Salmonella pathogenicity island l, that functions primarily to translocate effector proteins into the cytosol of host cells. These secreted effector proteins have cellular activities that induce uptake of the bacteria into intestinal cells. In addition, the effector proteins have activities that induce fluid secretion and inflammation within the small intestine that are hallmarks of enterocolitis. The expression of the type III secretion system and the associated effector proteins is dependent upon the HilA transcriptional activator, which is a member of the OmpR-ToxR family of transcriptional activators. Overexpression of the hilA gene results in a hyperinvasive phenotype while S. typhimurium hilA mutants are up to 500-fold less invasive than wild type strains for tissue culture cells, are significantly impaired in their ability to invade M cells of murine Peyer's patches and have oral LDs0 values for mice approximately 60-fold higher than the parent strain. The expression of hilA (and the type III secretion system that hilA activates) is regulated by a number of environmental signals (osmolarity, pH, oxygen, growth state) and genetic elements (hilC, hilD, sirA/barA, fliZ, phoBR, fadD, envZ, phoPQ, fis, arosE, pag and bilE). Our studies have focused on identifying and characterizing regulators of hilA expression. Recently, we found the fimZ response regulator activates a hilE-lacZY reporter. Subsequent work demonstrated that both the phoPQ two component regulator system and the phoBR two component system use the hilE/fimZ regulatory pathway to repress hilA expression. As a result, a variety of experiments are proposed to characterize those interactions in detail. In addition, we propose to identify genes regulated by the newly important FimZ response regulator, as well as genes that use FimZ to process signals from the environment. Finally, a previously reported pag repressor gene has been shown to have the ability to regulate the invasion transcriptional activator hilA within tissue culture cells. Experiments are proposed to characterize this repressor in detail as well. To determine the mechanisms by which these regulators affect hilA expression and its associated virulence phenotypes the following specific aims are proposed: 1) Identification and characterization of the signaling pathways that translate environmental signals into alterations in invasion gene expression. 2) Characterization of the FimZ regulon. 3) Identification and characterization of the pag gene and its role in intracellular hilA expression. The overall goal of this proposal is to understand the environmental signals and regulatory mechanisms that control expression of the SPI-1 hiIA activator, and the virulence mechanisms that hilA controls including invasion and gastroenteritis. An understanding of this regulatory system will lead to a better understanding of the complex interactions between this pathogen and the host and may lead to the identification of new therapeutic targets to control salmonellosis.