C-DI-GMP Signaling in E. Coli O157:H7 Biofilm Formation and Colonization of the Gastrointestinal Tract of Beef Cattle

Non-Technical Summary: E. coli O157:H7 induces huge losses to the meat industry and is a threat to consumer safety. Beef product recalls due to E. coli O157:H7 contamination are frequently associated with millions of pounds of product. Further, the runoffs from cattle farms can contaminate vegetables as shown in the 2006 nationwide spinach recall due to E. coli O157: H7 contamination. Since fecal shedding of E. coli O157:H7 from beef is the major source of this pathogen, it is important to reduce E. coli O157:H7 shedding in beef cattle, which depends on the reduction in E. coli O157:H7 gut colonization. On the other hand, biofilms on facility surfaces in processing plants provide a constant source of pathogens, including E. coli O157:H7. Once formed, the biofilm is very difficult to remove; it is resistant to washing and rinsing, chemical detergents and sanitizers. Its presence on facility surfaces and processing environment is a sustained source of contamination. Subsequently, prevention of biofilm formation is another key to beef safety improvement. However, up to now, our knowledge about the mechanisms regulating 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 pathogens, c-di-GMP signaling is likely involved in E. coli O157:H7 biofilm formation in food processing plants and in cattle gut colonization, which will be tested in the proposed studies. Knowledge obtained from this and subsequent studies will allow us to develop strategies to reduce E. coli O157:H7 contamination in beef and other foods. <P> Approach: To test our hypothesis that the c-di-GMP signaling regulates E. coli O157:H7 biofilm formation and gut colonization in beef cattle, we have two specific aims: 1) To assess the role of intracellular c-di-GMP level in E. coli O157:H7 biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) and is degraded by phosphodiesterase (PDE). We will transfect E. coli O157:H7 with constructed plasmids over-expressing DGC and PDE, and compare these with wild-type E. coli O157:H7 or E. coli O157:H7 carrying a control expression vector for their ability to form biofilms. In addition, we will examine the mRNA expression of E. coli O157:H7 carrying a control vector, as well as DGC and PDE expression vectors by microarray to identify genes differentially expressed; 2) To test the effects of intracellular c-di-GMP on E. coli O157:H7 colonization in epithelial cells and tissues. We will use in vitro cultured colon epithelial cells (HT-29), cattle colon primary epithelial cells and gut epithelial tissue explants for this study. We plan to use the constructed E. coli O157:H7 over-expressing DGC and PDE to alter the intracellular c-di-GMP level and to examine the role of c-di-GMP in E. coli O157:H7 colonization.