E. coli is the most common cause of community acquired urinary tract infection (UTI) and a leading cause of nosochomial UTIs and sepsis. There are an estimated 8 million physician visits per year in the U.S. for UTIs with significant associated morbidity and expense (>1,000,000,000). We tested the hypothesis that virulence genes responsible for the pathogenesis of life-threatening E. coli extraintestinal infections, such as pyelonephritis and sepsis can be identified by comparison of the genome sequence of urosepsis E. coli strain CFTO73 to either E. coli laboratory strain MG1655 or O157:H7 strain EDL933. We identified >300 CFT073-specific loci. The continued study of the region surrounding the D-serine deaminase genes (dsdCXA) is especially compelling. An allelic knockout mutant of dsdA which encodes D-serine deaminase, is unaltered in expression of type 1 pili-mediated adherence, but 300-fold more competitive than the wild type strain in colonizing the bladder or kidney of mice infected in an ascending model of UTI. In the future the Welch Lab will also identify environmental conditions and additional genes that affect the expression of the dsdCXA genes. The objective of the project is to identify and characterize critical virulence genes for E. coli involved in serious human diseases. This information will be of use for the development of new chemotherapeutic and vaccine strategies. Enterohemorrhagic Escherichia coli (EHEC), principally serotype O157:H7, cause an estimated 20,000 cases of diarrheal disease in the United States per year. 2-6% of the infected individuals, mostly young children progress to a severe renal disease, hemolytic uremic syndrome (HUS). The EHEC pathogenic factors that lead to bloody colitis and HUS are poorly understood, but knowledge of some mechanisms has recently emerged. Intimin-mediated adherence and type III effectors are encoded by a chromosomal locus termed LEE. The phage-encoded Shiga toxins (Stxs) are responsible for significant aspects of EHEC disease. EHEC strains commonly possess large plasmids, the prototype being pO157. We have identified a new pO157 gene, stcE, which encodes and extracellular zinc-metalloendoprotease that specifically cleaves the critical anti-inflammatory regulator C1-esterase inhibitor (C1-Inh). StcE was shown to contribute to intimate adherence of EHEC to host cells by cleavage of mucin-like glycoproteins from the enterocyte cell surface. Thus, StcE helps block host clearance of E. coli O157:H7 by destruction of some types of mucins, contributes to intimate adherence of E. coli O157:H7 and protects against complement lysis during the bloody colitis stage of its disease. The emerging picture of StcE protein substrates is that they are heavily glycosylated, mostly via O-linkages, and that the cleavage sites contain repeated amino acid sequences. Search is underway to define StcE’s activities toward different classes of mucins and mucin-like proteins on cell surfaces. The elucidation of StcE structure and function(s) may result in new targets for chemotherapeutic or immune prevention or treatment of EHEC infections, which now are best managed only by supportive therapy.