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.

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. 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

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Escherichia coli O157:H7

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