PCR versus hybridization for detecting virulence genes of enterohemorrhagic Escherichia coli.
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ABSTRACT:
SUBMITTER: Gerrish RS
PROVIDER: S-EPMC2828064 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In this report, we present a PCR protocol for rapid identification of enterohemorrhagic Escherichia coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min.
| S-EPMC87735 | biostudies-literature
Project description:Enteric pathogens with low infectious doses rely on the ability to orchestrate the 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 the expression of type III secretion system (T3SS) components to nutrient availability. While phosphorylation of histidine and aspartate residues on two-component system response regulators is recognized as an integral part of bacterial signaling, the involvement of phosphotyrosine-mediated control is minimally explored in Gram-negative pathogens. Our recent phosphotyrosine profiling study of E. coli identified 342 phosphorylated proteins, indicating that phosphotyrosine modifications in bacteria are more prevalent than previously anticipated. The present study demonstrates that tyrosine phosphorylation of a metabolite-responsive LacI/GalR family regulator, Cra, negatively affects T3SS expression under glycolytic conditions that are typical for the colonic lumen environment where production of the T3SS is unnecessary. Our data suggest that Cra phosphorylation affects T3SS expression by modulating the expression of ler, which encodes the major activator of EHEC virulence gene expression. Phosphorylation of the Cra Y47 residue diminishes DNA binding to fine-tune the expression of virulence-associated genes, including those of the locus of enterocyte effacement pathogenicity island that encode the T3SS, and thereby negatively affects the formation of attaching and effacing lesions. Our data indicate that tyrosine phosphorylation provides an additional mechanism to control the DNA binding of Cra and other LacI/GalR family regulators, including LacI and PurR. This study describes an initial effort to unravel the role of global phosphotyrosine signaling in the control of EHEC virulence potential.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) causes outbreaks of hemorrhagic colitis and the potentially fatal hemolytic-uremic syndrome. Successful host colonization by EHEC relies on the ability to coordinate the expression of virulence factors in response to environmental cues. A complex network that integrates environmental signals at multiple regulatory levels tightly controls virulence gene expression. We demonstrate that EHEC utilizes a previously uncharacterized phosphotyrosine signaling pathway through Cra to fine-tune the expression of virulence-associated genes to effectively control T3SS production. This study demonstrates that tyrosine phosphorylation negatively affects the DNA-binding capacity of Cra, which affects the expression of genes related to virulence and metabolism. We demonstrate for the first time that phosphotyrosine-mediated control affects global transcription in EHEC. Our data provide insight into a hitherto unexplored regulatory level of the global network controlling EHEC virulence gene expression.
| S-EPMC5829826 | biostudies-literature
Project description:The biogeography of the gut is diverse in its longitudinal axis, as well as within specific microenvironments. Differential oxygenation and nutrient composition drive the membership of microbial communities in these habitats. Moreover, enteric pathogens can orchestrate further modifications to gain a competitive advantage toward host colonization. These pathogens are versatile and adept when exploiting the human colon. They expertly navigate complex environmental cues and interkingdom signaling to colonize and infect their hosts. Here we demonstrate how enterohemorrhagic Escherichia coli (EHEC) uses three sugar-sensing transcription factors, Cra, KdpE, and FusR, to exquisitely regulate the expression of virulence factors associated with its type III secretion system (T3SS) when exposed to various oxygen concentrations. We also explored the effect of mucin-derived nonpreferred carbon sources on EHEC growth and expression of virulence genes. Taken together, the results show that EHEC represses the expression of its T3SS when oxygen is absent, mimicking the largely anaerobic lumen, and activates its T3SS when oxygen is available through Cra. In addition, when EHEC senses mucin-derived sugars heavily present in the O-linked and N-linked glycans of the large intestine, virulence gene expression is initiated. Sugars derived from pectin, a complex plant polysaccharide digested in the large intestine, also increased virulence gene expression. Not only does EHEC sense host- and microbiota-derived interkingdom signals, it also uses oxygen availability and mucin-derived sugars liberated by the microbiota to stimulate expression of the T3SS. This precision in gene regulation allows EHEC to be an efficient pathogen with an extremely low infectious dose.ImportanceEnteric pathogens have to be crafty when interpreting multiple environmental cues to successfully establish themselves within complex and diverse gut microenvironments. Differences in oxygen tension and nutrient composition determine the biogeography of the gut microbiota and provide unique niches that can be exploited by enteric pathogens. EHEC is an enteric pathogen that colonizes the colon and causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome worldwide. It has a very low infectious dose, which requires it to be an extremely effective pathogen. Hence, here we show that EHEC senses multiple sugar sources and oxygen levels to optimally control the expression of its virulence repertoire. This exquisite regulatory control equips EHEC to sense different intestinal compartments to colonize the host.
| S-EPMC5120142 | biostudies-literature
Project description:ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a principally foodborne pathogen linked to serious diseases, including bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Comparative genomics analysis revealed that EHEC O157 contains 177 unique genomic islands, termed O islands, compared with the nonpathogenic E. coli K-12 laboratory strain. These O islands contribute largely to the pathogenicity of EHEC O157:H7 by providing numerous virulence factors, effectors, virulence regulatory proteins, and virulence regulatory sRNAs. The present review aimed to provide a comprehensive understanding of the research progress on the function of O islands, especially focusing on virulence-related O islands.
| S-EPMC8565820 | biostudies-literature
Project description:Bacteriophage-encoded genetic elements control bacterial biological functions. Enterohemorrhagic Escherichia coli (EHEC) strains harbor lambda-phages encoding the Shiga-toxin (Stx), which is expressed during the phage lytic cycle and associated with exacerbated disease. Phages also reside dormant within bacterial chromosomes through their lysogenic cycle, but how this impacts EHEC virulence remains unknown. We find that during lysogeny the phage transcription factor Cro activates the EHEC type III secretion system (T3SS). EHEC lambdoid phages are lysogenic under anaerobic conditions when Cro binds to and activates the promoters of T3SS genes. Interestingly, the Cro sequence varies among phages carried by different EHEC outbreak strains, and these changes affect Cro-dependent T3SS regulation. Additionally, infecting mice with the related pathogen C. rodentium harboring the bacteriophage cro from EHEC results in greater T3SS gene expression and enhanced virulence. Collectively, these findings reveal the role of phages in impacting EHEC virulence and their potential to affect outbreak strains.
| S-EPMC5982111 | biostudies-literature
Project description:To better understand the role of host cell-derived molecules on enterohemorrhagic Escherichia coli (EHEC) infection, we studied EHEC virulence gene expression when exposed to cell-free spent (conditioned) medium (CM) from HCT-8 intestinal epithelial cells. Exposure to HCT-8 CM for 1 h and 3 h increased the expression of 32 of 41 EHEC locus of enterocyte effacement (LEE) virulence genes compared with fresh medium (FM). Expression of the Shiga toxin 1 (stx1B) gene was up-regulated at 1 h of exposure. Seventeen genes encoded by prophage 933W, including those for Stx2, were also up-regulated at both time-points. The increase in 933W prophage expression was mirrored by a 2.7-fold increase in phage titers. Consistent with the increase in virulence gene expression, we observed a fivefold increase in EHEC attachment to epithelial cells when exposed to CM. The increase in EHEC attachment was abolished when CM was heated to 95 °C or treated with proteinase K to degrade the proteins. The host cell-derived molecule(s) were larger than 3 kDa, which suggests that the molecule(s) that increase EHEC virulence and attachment are protein-based.
| S-EPMC3561499 | biostudies-literature
Project description:Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC encodes the sRNA chaperone Hfq, which is important in posttranscriptional regulation. In EHEC strain EDL933, Hfq acts as a negative regulator of the locus of enterocyte effacement (LEE), which encodes most of the proteins involved in type III secretion and attaching and effacing (AE) lesions. Here, we deleted hfq in the EHEC strain 86-24 and compared global transcription profiles of the hfq mutant and wild-type (WT) strains in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler, the transcriptional activator of all the LEE genes, as well as genes encoded in the LEE2 to -5 operons. Decreased expression of the LEE genes in the hfq mutant occurred at middle, late, and stationary growth phases. We also confirmed decreased regulation of the LEE genes by examining the proteins secreted and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC, which is involved in interkingdom signaling and virulence gene regulation in EHEC, as well as an increase in expression of stx(2AB), which encodes the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a regulatory role in EHEC 86-24 that is different from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE.
| S-EPMC3232842 | biostudies-literature
Project description:Gastrointestinal (GI) bacteria sense diverse environmental signals as cues for differential gene regulation and niche adaptation. Pathogens such as enterohemorrhagic Escherichia coli (EHEC), which causes bloody diarrhea, use these signals for the temporal and energy-efficient regulation of their virulence factors. One of the main virulence strategies employed by EHEC is the formation of attaching and effacing (AE) lesions on enterocytes. Most of the genes necessary for the formation of these lesions are grouped within a pathogenicity island, the locus of enterocyte effacement (LEE), whose expression requires the LEE-encoded regulator Ler. Here we show that growth of EHEC in glycolytic environments inhibits the expression of ler and consequently all other LEE genes. Conversely, growth within a gluconeogenic environment activates expression of these genes. This sugar-dependent regulation is achieved through two transcription factors: KdpE and Cra. Both Cra and KdpE directly bind to the ler promoter, and Cra's affinity to this promoter is catabolite dependent. Moreover, we show that the Cra and KdpE proteins interact in vitro and that KdpE's ability to bind DNA is enhanced by the presence of Cra. Cra is important for AE lesion formation, and KdpE contributes to this Cra-dependent regulation. The deletion of cra and kdpE resulted in the ablation of AE lesions. One of the many challenges that bacteria face within the GI tract is to successfully compete for carbon sources. Linking carbon metabolism to the precise coordination of virulence expression is a key step in the adaptation of pathogens to the GI environment. IMPORTANCE An appropriate and prompt response to environmental cues is crucial for bacterial survival. Cra and KdpE are two proteins found in both nonpathogenic and pathogenic bacteria that regulate genes in response to differences in metabolite concentration. In this work, we show that, in the deadly pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7, which causes bloody diarrhea, these two proteins influence important virulence traits. We also propose that their control of one or more of these virulence traits is due to the direct interaction of the Cra and KdpE proteins with each other, as well as with their DNA targets. This work shows how EHEC coopts established mechanisms for sensing the metabolites and stress cues in the environment, to induce virulence factors in a temporal and energy-efficient manner, culminating in disease. Understanding how pathogens commandeer nonpathogenic systems can help us develop measures to control them.
| S-EPMC3482499 | biostudies-literature
Project description:We have determined that the genes encoding the secreted proteins EspA, EspD, and EspB of enterohemorrhagic Escherichia coli (EHEC) are organized in a single operon. The esp operon is controlled by a promoter located 94 bp upstream from the ATG start codon of the espA gene. The promoter is activated in the early logarithmic growth phase, upon bacterial contact with eukaryotic cells and in response to Ca2+, Mn2+, and HEPES. Transcription of the esp operon seems to be switched off in tightly attached bacteria. The activation process is regulated by osmolarity (induction at high osmolarities), modulated by temperature, and influenced by the degree of DNA supercoiling. Transcription is sigmaS dependent, and the H-NS protein contributes to its fine tuning. Identification of the factors involved in activation of the esp operon and the signals responsible for modulation may facilitate understanding of the underlying molecular events leading to sequential expression of virulence factors during natural infections caused by EHEC.
| S-EPMC93807 | biostudies-literature
Project description:In E. coli the phosphate homeostasis is regulated by the Pst system and the two-component system PhoB/R. Pathogens like E. coli O157:H7 are responsible for many outbreaks and can be found and survive in poor inorganic phosphate (Pi) environments. To understand global EHEC O157:H7 EDL933 strain responses to Pi-starvation, we compared the transcriptomes of EDL933 the WT strain grown in MOPS Pi rich medium and that grown in MOPS Pi poor medium, using the Affymetrix GeneChip® E. coli Genome 2.0 Array. Also we investigated the EDL933 global response to the absence of PhoB by comparing the transcriptomes of the WT strain the ΔphoB mutant both grown in Low Pi EDL933WT strain and its isogenic mutant ΔphoB were grown in Pi- and/or in Pi+ conditions until the OD600 reached respectively 0.55 to 0.6. Samples equal to 5.108 CFU were taken from this mid log phase and processed for transcriptomes analysis. Ten µg of RNAs were extracted and retro-transcripted. One µg of the fragmented and biotinylated cDNAs, were hybridized onto Affymetrix GeneChip® E. coli. The data were processed using the FlexArray® software; RMA normalization and the levels of transcription obtained from 3 biological replicates of each experimental condition were compared using the EB (Wright & Simon) algorithm. We then conducted comparisons between EDL933WT grown in Pi- and in Pi+ conditions (comparison A) and between EDL933WT and EDL933∆phoB strains both grown in Pi- (comparison B). The differentially expression conditions corresponded to 2-fold change (FC) cut-off and p-value < 0.05.
2014-03-15 | E-GEOD-50529 | biostudies-arrayexpress