Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiling of 2 outbreak strains of E. coli O157:H7 following exposure to epithelial cells

ABSTRACT: Two outbreak strains of E. coli O157:H7 differ phylogenetically, in gene content, and in epidemiological characteristics. The working hypothesis in this experiment was that these strains will also differ in the transcription of shared virulence genes. Indeed, following a 30 minute exposure to epithelial cells, strain TW14359 overexpressed major and ancillary virulence genes, relative to strain Sakai. E. coli O157:H7 strains were physiologically normalized by growth to stationary phase, twice, in MOPS minimal media. Cultures were then transferred to DMEM media for adaptation. After 3 h of growth in DMEM, O157:H7 cultures were used to infect monolayers of MAC-T epithelial cells. 30 min following incubation, aliquots of suspended, non-adherent bacteria were used for RNA extraction. Five biological replications of the experiment were performed with each strain and, together with dye-swaps, 10 array hybridizations were carried out. Array data were fitted to a mixed model ANOVA using the following linear model: array+dye+sample (biological replicate)+strain+error.

ORGANISM(S): Escherichia coli

SUBMITTER: Galeb Abu-Ali

PROVIDER: E-GEOD-17389 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7.

Abu-Ali Galeb S GS Ouellette Lindsey M LM Henderson Scott T ST Whittam Thomas S TS Manning Shannon D SD

Microbiology (Reading, England) 20091105 Pt 2

The Escherichia coli O157 : H7 TW14359 strain was implicated in a multi-state outbreak in North America in 2006, which resulted in high rates of severe disease. Similarly, the O157 : H7 RIMD0509952 (Sakai) strain caused the largest O157 : H7 outbreak to date. Both strains were shown to represent divergent phylogenetic lineages. Here we compared global gene expression patterns before and after epithelial cell exposure, as well as the ability to adhere to and invade epithelial cells, between the t ...[more]

PMID: 19892762

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Project description:There is increasing evidence to support a role for sigma factor 54 (RpoN) in the regulation of stress resistance factors and protein secretion systems important to bacterial transmission and pathogenesis. In enterohemorrhagic E. coli O157:H7, acid resistance and type III secretion are essential determinants of gastric passage and colonization. This study thus described the transcriptome of an rpoN null strain of E. coli O157:H7 (EcJR-8) to determine the influence of RpoN on virulence and stress resistance gene regulation, and further explored its contribution to glutamate-dependent acid resistance (GDAR). Inactivation of rpoN resulted in the growth phase-dependent, differential expression of 104 genes. This included type III secretion structural and regulatory genes encoded on the locus of enterocyte effacement (LEE), as well as GDAR genes gadA, gadBC and gadE. Upregulation of gad transcript levels in EcJR-8 during logarithmic growth correlated with increased GDAR and survival in a model stomach. Acid susceptibility was reconstituted in EcJR-8 complemented in trans with wild-type rpoN. Acid resistance in EcJR-8 was dependent on exogenous glutamate, gadE and rpoS, but was independent of hns. Results also suggest that GDAR may be controlled by RpoN at multiple regulatory levels. This study supports the hypothesis that RpoN is an important regulator of virulence and stress resistance factors in E. coli O157:H7, and is the first to examine the mechanism by which it represses GDAR. Hybridizations measured transcriptional differences between an rpoN null and wild-type (WT) strain of E. coli O157:H7 Sakai at logarithmic and transition phase. Image files (TIFF) of hybridized microarray slides were generated using an Axon 4000B scanner (Molecular Devices), and analyzed using GenePix Pro software (Molecular Devices, ver. 6.0). The resulting microarray intensity data was log2-transformed, and normalized using the LOWESS algorithm in MAANOVA ver. 0.98-8 (R ver. 2.2.1).

Project description:Escherichia coli O157:H7 has caused serious outbreaks of foodborne illness via transmission in a variety of food vehicles, including unpasteurized apple juice, dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns after exposure to apple juice. Transcriptomes of mid-exponential and stationary phase cells were evaluated after 10 minutes in model apple juice (pH3.5) using microarrays probing 4,886 ORFs. Significant changes in gene expression were determined using R/MAANOVA and the Fs test. A total of 331 ORFs were significantly induced upon exposure of cells to model apple juice and included genes involved in the acid and osmotic stress responses as well as the oxidative stress response and envelope stress. Genes involved in the acid and osmotic stress responses, including asr, osmC, osmB, and osmY were significantly induced in response to model apple juice. Genes involved in the envelope stress response, known to be controlled by CpxR (cpxP, degP, and htpX), were significantly induced 2 to 15 fold upon exposure to apple juice, independent of growth phase. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 ORFs induced in model apple juice, 104 are O157-specific ORFs, including those encoding type three secretion effectors espJ, espB, espM2, espL3 and espZ. By elucidating the response of O157:H7 to acidic foods, we hope to gain insights into how this pathogen is able to survive in food matrices and how exposure to foods affects subsequent transmission and virulence. Keywords: stress The results are based on O157:H7 Sakai exponential and stationary phase cultures grown in MOPS minimal medium and then exposed to model apple juice (pH 3.5, 37C) for 10 minutes. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to growth phase (exponential or stationary), treatment (MOPS or MAJ) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+treatment+phase*treatment. We incorporated the dye-swaps among the biological replicates.

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Dataset Information

Transcriptional profiling of 2 outbreak strains of E. coli O157:H7 following exposure to epithelial cells

Publications

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7.

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