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Phosphotyrosine-mediated regulation of enterohemorhagic Escherichia coli virulence

ABSTRACT: Enteric pathogens with low infectious doses rely on the ability to orchestrate expression of virulence and metabolism-associated genes in response to environmental cues for successful infection. Accordingly, the human pathogen enterohemorrhagic Escherichia coli (EHEC) employs a complex multifaceted regulatory network to link expression of type III secretion system (T3SS) factors to nutrient availability. While phosphorylation of histidine and aspartate on two-component system response regulators is recognized as an integral part of signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli revealed 342 proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. Here, we demonstrate that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression in glycolytic conditions typical for the colon lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating expression of ler, encoding the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA-binding, thereby altering expression of metabolism and virulence-associated genes including those of the LEE pathogenicity island encoding the T3SS. Hence, phosphotyrosine-mediated regulation provides a mechanism to regulate Cra activity. Our data further suggest that tyrosine phosphorylation influences DNA binding by PurR and LacI, thereby phosphotyrosine-mediated control could provide a means to regulate DNA-binding of LacI/GalR family regulators in general. Our study provides an initial effort to unravel the role of phosphotyrosine-mediated global signaling in controlling the EHEC virulence potential

ORGANISM(S): Escherichia coli O157:H7 str. EDL933

PROVIDER: GSE103764 | GEO | 2018/02/02

REPOSITORIES: GEO

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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:EHEC is a food-borne pathogen that colonizes human GI tract and leads to infection. To understand the process of colonization and to decipher if any factors secreted by intestinal epithelial cells help EHEC during the infection process, we studied expression of EHEC virulence gene expression when exposed to intestinal epithelial cell conditioned medium. In our study, we observed that exposure to epithelial cell conditioned medium for 1 h and 3 h increases expression of 32 out of 41 EHEC LEE virulence genes. In addition, expression of the shiga toxin 1 (Stx1) gene is up-regulated at 1 h of exposure. Also, 17 genes encoded by prophage 933W, including those for Stx2, are also upregulated at both time-points. The increase in 933W prophage expression is mirrored by a 2.7-fold increase in intracellular Stx2 phage titers. Consistent with the increase in virulence gene expression, we observed a 5-fold increase in EHEC attachment to epithelial cells when exposed to conditioned medium, suggesting that EHEC utilizes host cell molecules to increase virulence and infectivity. The molecule(s) responsible for increased EHEC virulence is heat-sensitive as heating the conditioned medium to 95oC abolishes the increase in attachment to epithelial cells. A similar decrease was observed when the conditioned medium was treated with proteinase-K to degrade the proteins. The secreted molecule(s) was found to be larger than 3 kDa and strongly suggests that the HCT-8 secreted molecule that increases EHEC virulence and colonization is a protein-based molecule. Affymetrix E. coli Genome 2.0 Arrays were used to determine the changes in EHEC virulence gene expression on exposure to intestinal epithelial cell-secreted factors (through growth in conditioned medium). Overnight cultures of EHEC were diluted in LB medium to a turbidity of 0.1 at 600 nm. The cells were allowed to grow to a turbidity of 1.0 at 600 nm at 37Â°C and the EHEC cells were then resuspended in either fresh or conditioned medium. The cultures were then allowed to grow for 1 h or 3 h before cell pellets were collected by centrifugation and stored at -80Â°C. Total RNA was isolated from the cell pellets (173) and RNA quality was assessed using gel electrophoresis. Escherichia coli Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) containing 10,208 probe sets for all 20,366 genes present in four strains of E. coli, including EHEC, were used to profile changes in gene expression using RNA samples for each treatment.

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Phosphotyrosine-mediated regulation of enterohemorhagic Escherichia coli virulence

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