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This project promotes food safety through fundamental research applied to a specific agricultural problem: the ubiquitous presence of the human pathogen, Escherichia coli O157:H7 in food-producing ruminants. We will genetically characterize the ruminant: E. coli O157:H7 interaction using our expertise in molecular biology and our experience with experimental E. coli O157:H7 infections in adult cattle. The long-term goal of this proposal is to identify the genetic factors responsible for the persistence of E. coli O157:H7 in cattle. The proposed work tests our central hypothesis that specific gene expression is required for E. coli O157:H7 to colonize the bovine gastrointestinal tract. We will address two objectives:(1)to determine the role of virulence genes in E. coli O157:H7 colonization of the bovine recto-anal junction mucosa and (2) to determine differential gene expression in E. coli O157:H7 and bovine intestinal cells when the bacteria colonize the bovine recto-anal junction mucosa.
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It is well accepted that eliminating E. coli O157:H7 from cattle will reduce human foodborne illness with this microbe, but despite decades of research, an effective intervention to stop cattle from carrying E. coli O157:H7 is not known. Understanding the fundamental science that underlies the host:bacteria relationship is requisite to developing effective intervention strategies. The objective of this proposal is to increase our understanding of the food-borne pathogen E. coli O157:H7 and its relationship with cattle so that better intervention strategies can be developed. Our goal is to characterize the gene expression that allows E. coli O157:H7 to persist in cattle. The recognition of the bovine recto-anal junction mucosa as an E. coli O157:H7 colonization site, and the extraordinary situation that this site is easily accessible in live animals, has allowed us (with previous USDA-NRICGP funding) to develop a reproducible infection model, a culture technique that is superior to fecal culture, and to recover E. coli O157:H7 ex vivo without bacterial division in the laboratory. In this new work we will (1) determine if specific E. coli O157:H7 genes involved in human disease are also involved in colonization of cattle and, (2) use DNA microarray technology to study both bacterial and bovine gene expression during the host:bacteria interaction. </p>
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We will test isogenic derivatives that lack specific virulence genes or have been disabled in specific gene clusters to investigate the contribution of these factors to the bacteria's ability to colonization the bovine recto-anal junction(RAJ) mucosa. The derivatives will be engineered using the PCR-based one step gene inactivation system. We will test deletions in stx2, eae, tir, or ehxA that will remove the microbe's ability to synthesize the proteins Shiga toxin type 2, intimin, translocated intimin receptor, or entrohemolysin, respectively. </P>
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In addition, we will mutate ler, theLEE regulator and the lpf ABCC'DE gene cluster that encode long polar fimbriae that have been implicated in initial gastrointestinal attachment. Experimental cattle infections and monitoring for bacterial colonization will be done by a single rectal application of the bacteria and subsequent recto-anal mucosal swab sample bacterial culture. DNA microarray expression analysis will be used to measure alterations in bacterial and bovine gene expression at the RAJ. Ex vivo cells will be taken directly from this site. Steers will be infected by rectal application of E. coli O157:H7 and bacterial cells will be sampled from the recto-anal junction mucosa at time zero, 30 minutes, 1 hour, and 24 hours-post-inoculation. Total RNA will be extracted immediately, cDNA will be synthesized from the RNA with the incorporation of fluorescent dyes during reverse transcription using standard techniques. Purified labeled cDNA from experimental and reference samples (either time zero or laboratory grown bacteria) will be mixed in equal amounts and hybridized with the MWG E. coli O157 Array (MWG Biotech, High Point, NC, USA).</p>
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This microarray is a hybrid of 6,176 50-mer oligonucleotides and represents 5,326 genes in the E. coli O157:H7 EDL 933 genome. After overnight hybridization, the slides will be washed and analyzed with a confocal laser Genepix scanner. Similarly, bovine DNA microarrays will be used to measure the gene expression changes that follow E. coli O157:H7 colonization of the recto-anal junction mucosa, total RNA will be isolated from bovine cells scraped from the mucosal site over a time course following an experimental infection. Steer samples will be taken at time 0 hrs, 6 hrs, 1 day, 3 day, and 7 days. Messenger RNA will be captured and transcribed to cDNA, labeled with Cy-3 (green) or Cy-5 (red), and co-hybridized to the bovine microarray. The target set of genes is based on a 4500 gene bovine array from digestive tract, pituitary, and mammary gland bovine libraries. Comparisons among the hybridization reactions will be done in a simple loop design. K-means cluster analysis of gene changes over time will be determined. Lists of genes and their cellular function will be tabulated and analyzed. Affected biomarkers will be confirmed by Taqman (ABI Biosystems, Foster City, CA, USA) quantitative PCR.
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