Virulence gene regulators of enteric bacterial pathogens: Determining the structural and functional mechanisms of small molecule and polypeptide inhibitors

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Virulence gene regulators of enteric bacterial pathogens: Determining the structural and functional mechanisms of small molecule and polypeptide inhibitors Summary: The AraC/XylS family is one of the largest families of bacterial transcription factors with ~16K members distributed amongst 81% of sequenced bacterial species. Family members are present in pathogenic genera including Acinetobacter, Escherichia, Klebsiella, Legionella, Pseudomonas, Salmonella, Shigella, Vibrio, and Yersinia. The regulon of any given family member typically encompasses one of three categories: metabolism, stress response, or pathogenesis. Those involved in metabolism or stress response often have well characterized ligands. For example, AraC regulates the expression of genes involved in arabinose metabolism and its activity is modulated by arabinose. In contrast, small molecule ligands have not been identified for the vast majority of virulence regulators within the AraC family (hereafter referred to as AraC-VRs), which has led to the commonly held belief that the AraC-VR branch of the family has lost the ability to respond to ligands. Published work by us and others –and our preliminary studies– suggest that this assumption is incorrect. Additionally, a large family of endogenously encoded polypeptides, ANRs for AraC negative regulators, have been discovered that inhibit AraC-VRs though an unknown mechanism. The long-term goal of this project is to define the structural and molecular mechanisms underlying virulence gene regulation. The specific objectives of this proposal are to determine how AraC family members including Rns, a primary virulence regulator in enterotoxigenic E. coli (ETEC), are inhibited by 1) small molecule fatty acids and 2) AraC negative regulators (ANRs). Our central hypothesis is that Rns must dimerize in order to bind to DNA and regulate transcription and that these inhibitors block this by distinct mechanisms. The motivating rationale for these studies is that they will identify the molecular and structural requirements for inhibiting virulence gene expression, and will be tested by three specific aims: 1) Determine the structural mechanism by which ligand binding and dimerization regulates Rns activity; 2) Test our hypothesis that the Rns homolog RegA is regulated in the same manner; 3) Determine the mechanism by which ANRs inhibit Rns activity, and clarify if this is distinct from the inhibitory mechanism of small molecule fatty acids. This project is innovative in that the basic molecular mechanisms by which these proteins are regulated are not understood. Our multidisciplinary team, with expertise in microbiology, biochemistry, and structural biology, is uniquely positioned to undertake the proposed studies to determine these mechanisms. This research is significant, not only because it will answer outstanding questions of how AraC proteins function in enteric pathogens, but because we expect to demonstrate that AraC family proteins from a wide variety of enteric pathogens share a common mechanism of being inhibited by fatty acids. This will open up new possibilities for therapeutic strategies to combat global mortality and morbidity.