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Transcriptional Signatures of Salmonella Enteritidis Isolates with Varying Ability to Contaminate Intact Eggs


<p>I hypothesize that S. Enteritidis isolates that contaminate intact eggs have a unique transcriptional signature which differentiates them from the isolates that fail to contaminate intact eggs. I propose to test this hypothesis with the following specific aims:</p>

<li>Construct a gene expression microarray. A gene expression microarray will be constructed using oligonucleotide sequences of S. Enteritidis genes with single nucleotide polymorphisms (SNPs) that genetically differentiate egg contaminating isolates of S. Enteritidis from those of egg non-contaminating isolates.</li>
<li>Determine the differences in transcriptional signatures between S. Enteritidis isolates that differ in their ability to contaminate chicken eggs.</li>

<p>The differences between the transcriptomes of egg contaminating isolates of S. Enteritidis and those of egg non-contaminating isolates will be determined by gene expression microarray analysis. The next logical set of experiments, which will be the focus of the extramurally funded grant, will focus on determining and verifying function of the target genes (both in vitro and in vivo in a chicken infection model) by constructing gene knockout mutants and complementation in trans. The short term objective of this research is to produce preliminary data for the purpose of obtaining competitive grant funding to address my long-term objective to identify and characterize the functional aspects of S. Enteritidis genes that are expressed in the poultry environment as compared to genes that facilitate colonization of the hen and the completion of the infection pathway that results in egg contamination. Expected outputs: potential for extramural funding, research publication</p>

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<p>NON-TECHNICAL SUMMARY: Salmonella Enteritidis is one of the leading causes of food-borne gastroenteritis in humans. Epidemiological studies have frequently implicated S. Enteritidis contaminated chicken eggs as the major source of infections in humans. S. Enteritidis is a challenge to the safety of the food supply because this serotype can colonize the avian reproductive tract (RT), influence oviduct physiology, and subsequently contaminate internal content of intact eggs that are produced by otherwise healthy hens. It is now known that S. Enteritidis is phenotypically pleomorphic and that not all field isolates have the ability to complete the infection pathway that results in contamination of intact eggs; nevertheless, very little is know about the genetic factors that contribute to this effect. Therefore determining what genetic factors of S. Enteritidis facilitate colonization of the avian RT and the completion of the infection pathway resulting in egg contamination is critically important to devising improved detection, subtyping, and intervention methods and strategies to prevent or control this pathogen. Isolates of S. Enteritidis have been identified that are genetically related but that vary in terms of egg contamination in an in vivo infection model; these strains differ by a large number of single nucleotide polymorphisms (SNPs). The functional significance of these mutations (i.e. how they correlate with the differences in virulence factor expression in vitro or in vivo) remains to be fully ascertained. It is possible that the differences in the ability of S. Enteritidis isolates to infect hens and to subsequently contaminate intact eggs is a consequence of their altered transcriptional signatures due to the SNPs. By identifying unique transcriptional signatures for egg contaminating S. Enteritidis, we expect to clearly identify a group of genes and pathways that are characteristic to egg contaminating trait. This analysis will also provide a list of important targets for intervention or we will be able to infer important ecological differences that may provide clues to better intervention strategies.</p>

<p>APPROACH:<br />
Specific aim 1. Construction of a gene expression microarray.<br />
A gene expression microarray will be constructed using oligonucleotide sequences of S. Enteritidis genes with SNPs that genetically differentiate egg contaminating isolate of S. Enteritidis from that of egg non-contaminating isolate. The sequence information for a total of 120 target genes will be used for construction of microarray. Seventy-five additional genes will be included on the array because some of these have been recently reported to either contribute to the survival of S. Enteritidis in the internal contents of artificially inoculated eggs or are specifically induced during oviduct colonization or have been reported to play a role in virulence of S. Enteritidis. Two S. Enteritidis house keeping genes (eg., 16S rDNA and gyrB) will serve as positive controls. Two unique gene sequences from an unrelated organism (e.g., Flavobacterium psychrophilum) will be included on the array as negative controls. We will design 60 mer probes for each of the selected genes. Oligos will be printed onto Epoxysilane coated slides. The arrays' MIAME will be catalogued in the NCBI Gene Expression Omnibus.<br />
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Specific aim 2. Determine the differences in transcriptional signatures between S. Enteritidis strains that differ in their ability to contaminate chicken eggs.<br />
We will examine the gene expression profiles of 3 strains of S. Enteritidis that differ in their ability to contaminate intact eggs. One strain (PT13a21027) forms biofilm at 25C, does not produce HMM-LPS at 37C, and does not contaminate eggs. A second strain (PT13a 21046) does not form biofilm 25C, but produce HMM-LPS and contaminates eggs. The third strain is an isogenic strain of S. Enteritidis (PT4 22079) that produces biofilm at 25C, high HMM-LPS at 37C and contaminates hen egg after oral infection resulting in the recovery of only the wt S. Enteritidis subpopulation from eggs. Isolates will be inoculated into 10 ml brain heart infusion broth and incubated at for 24 h at 37C and 48 h at 25C without shaking. Bacterial cells will be pelleted and RNA will be extracted. cDNA will be generated following the recommended aminoallyl labeling procedure from PFGRC. The resultant aminoallyl-labeled cDNA will be chemically labeled using amine-reactive Alexa Fluor dye (A555 or A488). Spectrophotometry will be used to access dye incorporation per sample. Two target pools to be compared will be mixed prior to hybridization to the microarray slide. Hybridization procedures will follow the recommended protocols from PFGRC. Once hybridization procedures are complete, the slides will be scanned using an arrayWoRx scanner. Scanned slide images will be segmented using arrayTraker software. Subsequent quantitative data (median pixel values) will be downloaded to a customized rational database for data management and archiving. Our initial analysis will use Significance Analysis for Microarrays (SAM) software to compare transcription patterns between egg contaminating and egg-non contaminating S. Enteritidis grown under different conditions.</p>

Shah, Devendra
Washington State University
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