DESCRIPTION (provided by applicant): Campylobacter jejuni is a primary bacterial cause of gastroenteritis in the United States, with at least 2 million cases of C. jejuni gastroenteritis eah year in the U.S. (an incidence equal or greater to that of Salmonella and Shigella combined). C. jejuni is responsible for sporadic disease as well as food-borne / water- borne outbreaks, which result from contaminated food and exposure to recreational waters. Some C. jejuni infections lead to the development of Guillain-Barré Syndrome, the leading cause of acute paralysis in the world. Despite the high prevalence of Campylobacter disease and more than 20 years of study, the mechanisms by which C. jejuni causes disease remain incompletely understood and severely understudied. In many bacteria, CsrA regulates numerous important phenotypes, including virulence, carbon metabolism, motility, quorum sensing, biofilm production, and animal colonization. While in all Gram-negative bacteria studied to date CsrA activity is controlled by inducible small regulatory RNAs (sRNAs), new data suggests for the first time that C. jejuni CsrA is regulated by novel protein-protein interactions with FliW, a member of the flagellar biosynthesis pathway, thus linking the synthesis of the critical virulence factors flagella to the regulation of other stationary phase cell processes such as biofilm formation. We constructed a C. jejuni mutant lacking csrA, and have shown that the csrA mutant exhibits pleiotropic virulence-related phenotypes including decreased motility, epithelial cell adherence, biofilm formation, resistance to oxidative stress, and colonization of mice. Conversely, the csrA mutant shows increased invasion of human epithelial cells. This underscores the importance of the CsrA regulon in C. jejuni pathogenesis. We have determined the presumptive CsrA regulon, and it contains a number of proteins with clear links to flagellar motility / chemotaxis and to stationary phase processes such as biofilm formation and acetate metabolism. Finally, we have demonstrated direct interaction of FliW with CsrA, supporting the link between flagella and CsrA regulation. Overall hypothesis: C. jejuni CsrA plays an important role in the pathogenesis of C. jejuni via stationary phase, post-transcriptional regulation of virulence / survival properties including motility and biofilm formation. We propose a detailed study of CsrA-mediated post-transcriptional stationary phase gene regulation in C. jejuni, focusing on the mechanism by which CsrA controls the expression of motility and biofilm formation. We will use genetic, proteomic and biochemical approaches to achieve the goals outlined in these two specific aims: Aim 1) Define the roles of CsrA, FliW, and FlaA in coordinating stationary phase regulation of motility and chemotaxis, and Aim 2) Define the role of CsrA in regulating biofilm production and acetate metabolism.
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