1a. Objectives (from AD-416)
1) Determine and characterize molecular mechanisms promoting colonization, effective adherence, and persistence of E. coli O157:H7 and other STECs in cattle; 2) Understand the impact of bovine intestinal environment and immune responses on growth, adherence, and persistence of E. coli O157:H7 and other STECs in cattle; 3) Conduct comparative analysis of bovine E. coli O157:H7 and STEC isolates of public health significance to identify components for use in developing rapid diagnostic tools and effective interventions; and 4) Develop and test efficacy of chemical, biological, subunit proteins, and whole cell vaccines to prevent or reduce colonization of cattle intestines by E. coli O157:H7 and STECs.
1b. Approach (from AD-416)
Experimental animal models, tissue cultures, and specific mutants will be used to describe molecular mechanism(s) enabling E. coli O157:H7 bacteria to grow, adhere, and colonize the cattle intestine. Reporter gene fusions and global gene analysis technologies will be used to determine effects of host gastrointestinal environment and innate immune system on the expression of specific bacterial genetic systems and metabolic pathways that promote E. coli O157:H7 persistence in cattle intestine. Emerging non-O157 STEC serotypes will be compared with E. coli O157:H7 to identify genetic and molecular features unique to these serotypes. Bacterial genes or gene products identified in these studies will be used, based on their importance in colonization, for developing whole-cell or protein/subunit protein vaccines for reducing or eliminating E. coli O157:H7 and non-O157 STEC colonization and shedding in cattle.
3. Progress Report
Objective 1. Molecular mechanisms promoting colonization: a) Constructed some of the mutant strains of O157 that will facilitate understanding of the relative contributions of various bacterial cell surface structures in adherence of O157 to epithelial cells and in colonization of cattle intestines; b) Standardized the protocol for filter sterilizing rumen fluid from cattle on various diets. The sterilized rumen fluid was used to grow O157 strains under different growth conditions to determine genes expressed and to set up baseline for comparison with growth in vivo and in different media; c) Used squamous cells from recto-anal junctions to develop a novel adherence assay for evaluating the potential of O157 and non-O157 bacteria for their ability to adhere to recto-anal junction (RAJ); d) Characterized squamous epithelial cells to determine cell surface and genetic markers to set up baseline for experiments to immortalize these cell lines with the goal to have an indeterminate supply of these cells for performing adherence assays. Objective 2. Impact of bovine intestinal environment and immune responses on O157 and shiga toxin-producing Escherichia coli (STEC) persistence in cattle: a) Constructed O157 mutants that are needed to understand if mammalian stress hormones produced by intestinal tissues and bacterial chemical signaling molecules produced by the intestinal bacteria promote O157 colonization of cattle intestines.
1. Molecular mechanisms promoting colonization. Differences in growth patterns of Escherichia coli 0157 (E. coli O157) in cattle rumen fluid was found by ARS researchers at Ames, Iowa to be closely associated with differences in proteins expressed by this food-borne pathogen that is associated with cattle-derived food products. These proteins have the potential for use as vaccines to reduce the levels of E. coli O157 in intestines and feces of cattle, which in turn would reduce the risk of contamination meat products produced from these animals.
Kudva, I.T., Nystrom, E.A. 2011. Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence. Journal of Applied Microbiology. 111(5):1283-1294.