C-DI-GMP signaling in Escherichia Coli O157:H7 Biofilm Formation and Gastrointestinal Tract Colonization of Beef Cattle

NON-TECHNICAL SUMMARY: E. coli O157:H7 induces huge losses to the beef industry and is a threat to consumer safety. Fecal shedding of E. coli O157:H7 from beef cattle is the major source of this pathogen and biofilms on facility surfaces in processing plants provide a constant source of pathogens, including E. coli O157:H7. Up to now, our knowledge about the mechanisms regulating E. coli O157:H7 biofilm formation and gut colonization is very limited. The c-di-GMP molecule is a cytoplasmic second messenger that mediates a myriad of cellular processes. Based on its roles in regulating motility, biofilm formation and virulence gene expression in other pathogenic microorganisms, c-di-GMP signaling is likely involved in E. coli O157:H7 biofilm formation in food processing plants and in cattle gut colonization. Through studies proposed in this project, we will assess the role of c-di-GMP signaling in E. coli O157:H7 colonization and biofilm formation. As an extension of such studies, we will try to develop specific strategies to control E. coli O157:H7 colonization in gastro-intestine tract by targeting identified molecular mediators. Because c-di-GMP signaling is a universal signaling molecule and is involved in biofilm formation and virulence gene expression, knowledge obtained through these studies will have wide applications. These applications will not only be limited to E. coli O157:H7, but will deepen our understanding of food-borne pathogens and their pathogenesis in general, facilitating our efforts to provide consumers with safer foods.

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APPROACH: There are three specific aims: 1) To assess the role of intracellular c-di-GMP levels in E. coli O157:H7 global gene expression. We will achieve elevated (or lowered) levels of intracellular c-di-GMP by over-expressing diguanylate cyclase, DGC (c-di-GMP synthase) or phosphodiesterase, PDE (c-di-GMP hydrolase). To monitor gene expression, we will use oligo-based whole genome E. coli O157:H7 DNA microarrays and confirmed by quantitative real time PCR. 2) To test the c-di-GMP signaling on E. coli O157:H7 colonization and cytotoxicity to epithelial cells and tissues through in vitro epithelial cell line and primary cell culture using constructed plasmids expressing DGC or PDE. 3) To evaluate the functions of c-di-GMP in E. coli O157:H7 biofilm formation and colonization through major DGC and PDE gene deletions. To further test the possible role of intracellular c-di-GMP signaling in E. coli O157:H7 biofilm formation and gut epithelium interaction, we will delete selected endogenous DGC and PDE genes such as yeaI, yeaJ and ydiV, yhjH. The ability of biofilm formation and epithelium colonization and potential virulence of resulting mutated E. coli O157:H7 will be tested.