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 plasmids for complementation of the mutant strains of Escherichia coli (E. coli) O157:H7 to understand relative contributions of various bacterial cell surface structures and proteins in adherence of E. coli O157:H7 to epithelial cells and in colonization of cattle intestines; b) Used sterilized rumen fluid to grow non-O157:H7 strains under different growth conditions in order to set up baseline for comparison with growth in vivo and in vitro on different media; c) Recently standardized bovine recto-anal junction squamous epithelial (RSE) cell adherence assay was used to determine adherence potential and the adherence mechanisms used by a collection of O157:H7 and non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) strains to RSE; d) Performed adherence-inhibition assays by using sera directed to some of the known adhesins to determine if the observed adherence of O157:H7 mutants lacking expression of these adhesins is due to additional unknown adherence factors; Proteomic analysis was done to identify novel adhesins being expressed by O157:H7 that may have a role in the observed adherence to RSE cells. Objective 2. Impact of bovine intestinal environment and immune responses on E. coli O157:H7 and STEC persistence in cattle: a) Constructed E. coli O157:H7 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 E. coli O157:H7 colonization of cattle intestines. Objective 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: a) The immune response to a non-O157:H7 Escherichia coli vaccine was evaluated for cross-reactive antibodies to E. coli O157:H7.
Sharma, V.K., Sacco, R.E., Kunkle, R.A., Bearson, S.M., Palmquist, D.E. 2012. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle. Infection and Immunity. 80(4):1333-1342.