|Professor of Medical Microbiology & Immunology|
Chair · On sabbatical 2/1/13 - 7/31/13
|6157 Microbial Sciences Building|
1550 Linden Drive
|Office: (608) 263-2700|
1974 - B.S., Cornell University, Ithaca, NY
1979 - Ph.D., Medical College of Virginia, Richmond, VA
1980-81 - Postdoctoral fellowship, University of Washington, Seattle, WA
1981-82 - Postdoctoral fellowship, Stanford University, Stanford, CA
Project 1: Identification and Regulation of VirulenceFactors in Uropathogenic Escherichia coli
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 strin 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. DsdC is a positive effector of dsdXA transcription and a member of the lysR-family of regulators. By in vivo and in vitro gene expression techniques we tested the hypotheses that D-serine through interaction with either dsdC or other co-effectors affects expression of multiple genes that directly influence CFTO73 pathogenesis in murine models of disease. Through in vivo transcriptome analysis CFT073 dsdA mutant displays eleveated expression of >70 specific genes, many of which are known virulence factors. In the future the Welch Lab will also identify environmental conditions and additional genes that affect the expression of the dsdCXA genes. Two such gene candidates are ipuAB (integrase-like proteins of uropathogens) that are immediately adjacent to the dsdCXA genes in the chromosome of CFTO73 as well as other uropathogenic E. coli ipuAB are homologs of the type 1 pili phase-switch recombinases, fimB and fimE that are linked to and control expression of the E. coli type 1 pilus fim operon. 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.
Project 2: Enterohemorrhagic E. coli Pathogenesis
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). C1-Inh is a serine protease inhibitor (serpin) that provides the pricipal inhibition of the proteolytic cascades involved in classic and mannan-binding ligand complement activation, contact activation and intrinsic coagulation. C1-Inh inhibits diverse proteases: C1r and C1s, MASP-1, MASP-2, kallikrein, FXIIa, FXIa, and plasmin. Deficiencies in C1-Inh cause profound clinical syndromes. The best known is hereditary angioedema (HAE), a genetic deficiency in C1-Inh, which is characterized by transient recurrent attacks of intestinal cramps, vomiting, diarrhea and life-threatening episodes of tracheal swelling. Analyses of StcE-treated C1-INH activity revealed that, surprisingly, StcE enhanced the ability of C1-INH to inhibit the classical complement-mediated lysis of sheep erythrocytes. StcE directly interacts with both cells and C1-INH, thereby binding C1-INH to the cell surface. This suggests that the augmented activity of StcE-treated C1-INH is due to the increased concentration of C1-INH at the sites of potential lytic complex formation. We have recently found that StcE cleaves C1-Inh within a mucin-like domain. StcE does not act as a general protease and two new StcE substrates, mucin 7 and glycoprotein 340 were also recently discovered. 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.