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.
1. Molecular mechanisms promoting persistence and fecal shedding of Escherichia coli (E. coli) O157:H7 in cattle. An understanding of the mechanisms that promote colonization and increase fecal shedding of E. coli O157:H7 bacteria in cattle intestines are critical for developing vaccines to reduce and/or eliminate E. coli O157:H7 in cattle. Reduced fecal shedding of E. coli O157:H7 will lower the risk of contamination of hides and carcasses, which in turn will lower the risk for contamination of meats produced from these animals. By infecting cattle with specific E. coli O157:H7 variants capable of secreting specific proteins that allow adherence of these bacteria to the tissues of the large intestine, ARS researchers at Ames, IA found that these variants were shed in the feces for longer duration and at higher numbers in infected animals than those E. coli O157:H7 variants that were unable to secrete these proteins. The understanding of the requirement of this protein secretion mechanism and specific proteins secreted through this mechanism in colonization of cattle intestines has enabled construction and evaluation of novel heat-killed vaccines for reducing colonization and fecal shedding of E. coli O157:H7.
2. Bacterial proteins are required for adherence of Escherichia coli (E. coli) O157:H7 to cattle intestinal tissues. E. coli O157:H7 bacteria colonize terminal portion of the large intestine of cattle by adhering or “sticking” to a specific type of tissue or cells that make up this portion of the large intestine. Earlier studies have implicated one specific type of protein (intimin) responsible for adherence of E. coli O157:H7 to this particular tissue type of large intestine. However, ARS researchers at Ames, IA have determined that E. coli O157:H7 lacking the intimin protein uses additional unknown proteins for adherence to the other tissue types present in the terminal part of the large intestine. This finding is significant in the context of developing efficacious vaccines for blocking adherence of E. coli O157:H7 bacteria to cattle large intestines. Therefore, better vaccines would be the one that would include not only the intimin protein but other proteins, once identified, to reduce adherence of E. coli O157:H7 to all tissues in the terminal part of the large intestine.
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.
Casey, T., Connell, T.D., Holmes, R.K., Whipp, S.C. 2012. Evaluation of heat-labile enterotoxins type IIa and type IIb in the pathogenicity of enterotoxigenic Escherichia coli for neonatal pigs. Veterinary Microbiology. 3(18). Available: http://www.sciencedirect.com/science/article/pii/S037811351200185X.
Kudva, I.T., Griffin, R.W., Krastins, B., Sarracino, D.A., Calderwood, S.B., Manohar, J. 2012. Proteins other than the Locus of Enterocyte Effacement-encoded proteins may contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells. BMC Microbiology. 12(103). Available: http://www.biomedcentral.com/1471-2180/12/103.