Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Microarray Analysis of Acid-Stress Induced Changes in EHEC O157:H7

ABSTRACT: While significant advances have been made in EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. During the course of infection, EHEC must evade or overcome several biological barriers, the first of which is the gastric acidity encountered during passage through the stomach. EHEC is remarkable in its ability to tolerate this acidity. There are four different acid resistance systems that provide E. coli O157:H7 protection against exposure to low pH (2-2.5). Interestingly, EHEC uses these acid resistance systems differentially for survival in foods versus the bovine intestinal tract. The glutamate-dependent acid-resistance system is thought to offer the best protection below pH 3. Since the infectious dose of EHEC is so low (50-100 organisms), acid resistance becomes an important virulence trait. Studies of EHEC response to acid stress have focused primarily on levels of acid tolerance and the molecular basis of tolerance. However, the impact of acid stress on EHEC virulence is less well understood. In the related pathogen, EPEC, the plasmid-encoded regulator, Per, that regulates expression of many EPEC virulence factors, is regulated negatively at pH 5.5 and positively at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced in alkaline pH typical of the small intestine. Expression of EPEC type III secreted factors involved in A/E lesion formation has been shown to be influenced by factors including culture media, iron and calcium levels. Protein secretion was inhibited at pH 6 and 8. In a third study, a gadE (encoding acid resistance regulator) mutation resulted in increased adhesion of E.coli O157:H7 to colonic epithelial cells, suggesting negative regulation of one or more adhesins. Other studies have reported that shiga toxin production is sensitive to culture conditions including pH. However, there are no studies of EHEC virulence changes after more severe acid stress nor studies linking stressed EHEC virulence phenotype with transcriptional changes. The goal of this study was to determine how acid stress affects EHEC virulence properties and through microarray analysis, define the genetic basis for these changes. Understanding how acid stress modulates the virulence potential of this pathogen is essential for delineating the pathogenesis of disease caused by EHEC infection and may offer novel approaches to prevent and treat EHEC infections. Bacteria were grown in LB broth overnight, then subcultured into DMEM and grown at 37C, 5%Co2. Bacteria were then subjected to one of three acid stress protocols: 1) UA30: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 30 minutes; 2) AA30: growth in DMEM pH 5.0 (adaptation) followed by growth in DMEM pH 3.0; 3) UA15: growth in DMEM pH 7.4 followed by growth in DMEM pH 3.0 for 15 minutes. DMEM was supplemented with 25 mM MES (pH 5.0) and in the case of the control (unadapted, unshocked) 25 mM MOPS (pH 7.4) and the adaptation step was again carried out at 37C and 5% CO2. Acid shocking was done at pH 3.0 (unbuffered) at room temperature for all treatments

ORGANISM(S): Escherichia coli

SUBMITTER: Julianne Kus

PROVIDER: E-GEOD-14069 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence.

House B B Kus J V JV Prayitno N N Mair R R Que L L Chingcuanco F F Gannon V V Cvitkovitch D G DG Barnett Foster D D

Microbiology (Reading, England) 20090604 Pt 9

Enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 is naturally exposed to a wide variety of stresses including gastric acid shock, and yet little is known about how this stress influences virulence. This study investigated the impact of acid stress on several critical virulence properties including survival, host adhesion, Shiga toxin production, motility and induction of host-cell apoptosis. Several acid-stress protocols with relevance for gastric passage as well as external environmental ex ...[more]

PMID: 19497950

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Project description:Enterohemorrhagic Escherichia coli (EHEC), including serotype O157:H7, cause severe food-borne illness. On route to the human colon, they encounter and resist, numerous anti-microbial ingestion stresses. We hypothesize that these stresses cue EHEC to alter virulence properties. This study investigated the impact of bile salts on virulence properties and examined the genetic basis of the phenotypes. Established assays were used to examine adhesion to human epithelial cells, motility, verotoxin (VT) production and antimicrobial resistance with/without bile salt stress. Bacteria treated for 90 minute in DMEM plus 0.15% (w/v) bile salt mix demonstrated significantly enhanced adhesion to epithelial cells and resistance to several antibiotics but did not increase motility or VT production. To determine the genetic basis of these phenotypes a microarray experiment was conducted. EHEC strain 86-24, in mid-log phase of growth, were grown in DMEM pH 7.4 (control), or DMEM plus bile salt mix (0.15% w/v), for 90 minutes, statically at 37˚C, 5% CO2 prior to harvesting RNA for the microarray study. Four biological replicates were produced for each treatment. Microarray and gene expression analysis (semi-quantitative RT-PCR and beta-galactosidase reporter assays) of bile salt-treated EHEC revealed significant up-regulation of genes for lipid A modification, fimbriae, an efflux pump, and a two-component regulatory system relative to the bacteria grown in DMEM alone. This work points to several mechanisms that EHEC employs to resist the stresses of the human small intestine, notably efflux, antimicrobial resistance, and outer membrane alterations. Bile salts enhanced the virulence-related properties of increased adhesion and resistance to antimicrobials but not VT production or motility. This research contributes to our understanding of how EHEC senses and responds to host environmental signals and the mechanisms this pathogen uses to successfully colonize and infect the human host. Bacteria were grown in LB broth overnight with shaking, then subcultured into DMEM and grown statically at 37˚C, 5%CO2 to mid-log phase. Bacteria were then subjected to one of two 90 minute treatments: 1) Control: DMEM pH 7.4, or 2) Bile Salt Stress: DMEM pH 7.4 plus 0.15%, grown statically at 37˚C, 5%CO2.

Project description:While significant advances have been made in EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. During the course of infection, EHEC must evade or overcome several biological barriers, the first of which is the gastric acidity encountered during passage through the stomach. EHEC is remarkable in its ability to tolerate this acidity. There are four different acid resistance systems that provide E. coli O157:H7 protection against exposure to low pH (2-2.5). Interestingly, EHEC uses these acid resistance systems differentially for survival in foods versus the bovine intestinal tract. The glutamate-dependent acid-resistance system is thought to offer the best protection below pH 3. Since the infectious dose of EHEC is so low (50-100 organisms), acid resistance becomes an important virulence trait. Studies of EHEC response to acid stress have focused primarily on levels of acid tolerance and the molecular basis of tolerance. However, the impact of acid stress on EHEC virulence is less well understood. In the related pathogen, EPEC, the plasmid-encoded regulator, Per, that regulates expression of many EPEC virulence factors, is regulated negatively at pH 5.5 and positively at pH 8.0, suggesting that virulence gene expression is repressed during mild acid stress and enhanced in alkaline pH typical of the small intestine. Expression of EPEC type III secreted factors involved in A/E lesion formation has been shown to be influenced by factors including culture media, iron and calcium levels. Protein secretion was inhibited at pH 6 and 8. In a third study, a gadE (encoding acid resistance regulator) mutation resulted in increased adhesion of E.coli O157:H7 to colonic epithelial cells, suggesting negative regulation of one or more adhesins. Other studies have reported that shiga toxin production is sensitive to culture conditions including pH. However, there are no studies of EHEC virulence changes after more severe acid stress nor studies linking stressed EHEC virulence phenotype with transcriptional changes. The goal of this study was to determine how acid stress affects EHEC virulence properties and through microarray analysis, define the genetic basis for these changes. Understanding how acid stress modulates the virulence potential of this pathogen is essential for delineating the pathogenesis of disease caused by EHEC infection and may offer novel approaches to prevent and treat EHEC infections.

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.

Dataset Information

Microarray Analysis of Acid-Stress Induced Changes in EHEC O157:H7

Publications

Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157 : H7 virulence.

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