2010 Annual Report
1a.Objectives (from AD-416)
1) Describe and characterize sites, mechanisms, and Stx-mediated effects of STEC colonization of cattle intestines;.
2)Analyze in vivo gene expression of STEC O157:H7 using genomic techniques to identify genes involved in expression and regulation of adherence, colonization, and shedding;.
3)Develop and test the efficacy of intervention strategies for reducing colonization and shedding of STEC O157:H7; and.
4)Identify mechanisms of E. coli adherence involved in postweaning colibacillosis in pigs.
1b.Approach (from AD-416)
Experimental animal infections and genomic technologies will be used to identify specific STEC genes necessary for colonization and persistence in animals, and to identify and test interventions directed against identified targets. Weaned calves and neonatal pigs will be experimentally inoculated with E. coli. Microbiologic, histologic, and immunologic methods will be used to identify colonization sites and mechanisms of host-pathogen interactions. Biochemical, immunologic, and molecular biologic techniques will be used to identify specific STEC genes necessary for colonization and persistence in animals, and to identify and test interventions (vaccines and antimicrobials) directed against identified targets. Genetic, molecular, and immunologic techniques will be used to identify and characterize bacterial adhesins and other virulence factors of E. coli pathogens which cause diarrhea and edema disease in postweaning swine and evaluate their usefulness as targets for diagnostic assays or immunogens to prevent colibacillosis in swine.
Escherichia coli (E. coli) O157 Colonization: Demonstrated that O157 bacteria deleted of a master regulator gene, called hha, showed enhanced adherence to epithelial cells in tissue culture assays. We further demonstrated that O157 lacking the master regulator showed reduced motility but increased expression of genes required for the synthesis of cell surface molecules (intimin, EspA, and curli) promoting adherence of O157 to epithelial cells. These affects of master regulator hha on motility and adherence were independent of a mechanism that controls motility and adherence via sensing the population density (quorum sensing) of O157 bacteria. These results suggest that interactions between multiple gene expression pathways determine the ability of O157 to colonize and persist in cattle intestines. Shiga toxin-producing Escherichia coli (STEC) Colonization: Developed and standardized for the first time laboratory protocols for the isolation and growth in laboratory cultures of specific cell types (squamous cells) recovered from the terminal large intestine (recto-anal junction or RAJ) of cattle. Since these cell-types have been shown to serve as a major site for O157 colonization in cattle, availability of the developed protocols will facilitate rapid determination of the patterns and the mechanisms responsible for interactions between O157 and squamous cells without resorting to the use of live cattle. Interventions: Constructed genetic mutants of O157 that showed increased expression of cell surface molecules considered important for adherence of O157 bacteria to cattle intestines. Increased expression of some of these surface molecules was confirmed by antibody-based protein detection assays. Our goal is to use O157 bacteria expressing increased levels of these surface molecules as heat-killed vaccines for reducing O157 colonization and persistence in cattle. Inventions: A small, non-antibiotic, metabolic inhibitor was found to prevent the growth of O157 E. coli in vitro. In a limited, preliminary trial, this inhibitor reduced O157 fecal shedding following experimental infection in weaned calves. These early results are encouraging and suggest that this inhibitor may be useful as a pre-harvest intervention for decreasing or preventing O157 infections in cattle.
Describing the Mechanism that Promotes Adherence of O157:H7 to Epithelial Cells. An understanding of the mechanism that promotes adherence of O157:H7 to epithelial cells can provide valuable information with regards to genes and proteins essential for O157:H7 colonization of bovine intestines. By comparing two mutant strains with the parent strain of O157:H7 in adherence assays, ARS researchers at Ames, Iowa found that increased motility was detrimental to adherence while increased expression of specific structures and other molecules on bacterial cell surface increased adherence to epithelial cells. This work is a key to the understanding of the role of various cell surface molecules in colonization and/or long-term persistence of O157:H7 in cattle intestines and to the potential use of these molecules in whole-cell vaccines for reducing O157:H7 colonization and persistence in cattle.
Sharma, V.K., Bearson, S.M., Bearson, B.L. 2010. Evaluation of the Effects of SDIA, a LUXR Homologue, on Adherence and Motility of Escherichia coli O157:H7. Microbiology. 156(5):1303-1312.
Charles, R.C., Sheikh, A., Krastins, B., Harris, J., Bhuiyan, S.M., Larocque, R.C., Logvinenko, T., Sarracino, D.A., Kudva, I.T., Eisenstein, J., Podolsky, M.J., Kalsy, A., Ludwig, A., John, M., Calderwood, S.B., Qadri, F., Ryan, E.T. 2010. Characterization of Anti-Salmonella enterica Serotype Typhi Antibody Responses in Bacteremic Bangladeshi Patients by an Immuno-affinity Proteomic-based Technology (IPT). Clinical and Vaccine Immunology. 17:1188-1195.