EDL933 Strains of Escherichia coli O157 can Demonstrate Genetic Diversity and Differential Adherence to Bovine Recto-Anal Junction Squamous Epithelial Cells

Background: Differences between Escherichia coli O157 (O157) strains are well-established with some of these strains being associated with major outbreaks in the US. EDL933 is one such O157 strain that caused a multistate outbreak in 1982 and has since been used as a prototype in various O157-related experiments. Objective: As O157 can readily acquire genetic mutations, we sought to determine if the genetic and phenotypic profiles of EDL933 strains from different sources would be consistent. Methods: We evaluated wild-type O157 strains stocked as EDL933 from three different laboratories, in the strain typing Polymorphic Amplified Typing Sequence (PATS) and the bovine rectal-anal junction squamous epithelial (RSE) cell- and HEp-2 cell- adherence assays. In addition, we also verified if Shiga toxins (Stx), the Locus of Enterocyte Effacement (LEE) or curli fimbriae contributed to the adherence phenotypes observed using mutant and wild-type EDL933 isolates. Results: Our results showed differences in PATS profiles and RSE cell-adherence phenotype, with no influence from the Stx or LEE genes, between EDL933 from different sources. Interestingly, the EDL933 strain that demonstrated the most contrasting diffuse adherence phenotype on RSE cells, EDL933-T, had decreased curli production that may have contributed to this phenotype. Conclusion: Our observations suggest that a comprehensive characterization of bacterial isolates, even if assigned to the same strain type prior to use in experiments, is warranted to ensure consistency and reproducibility of results.

The transcriptional activator of the bfp operon in EPEC (PerA) interacts with the RNA polymerase alpha subunit

Enteropathogenic E. coli virulence genes are under the control of various regulators, one of which is PerA, an AraC/XylS-like regulator. PerA directly promotes its own expression and that of the bfp operon encoding the genes involved in the biogenesis of the bundle-forming pilus (BFP); it also activates PerC expression, which in turn stimulates locus of enterocyte effacement (LEE) activation through the LEE-encoded regulator Ler. Monomeric PerA directly binds to the per and bfp regulatory regions; however, it is not known whether interactions between PerA and the RNA polymerase (RNAP) are needed to activate gene transcription as has been observed for other AraC-like regulators. Results showed that PerA interacts with the alpha subunit of the RNAP polymerase and that it is necessary for the genetic and phenotypic expression of bfpA. Furthermore, an in silico analysis shows that PerA might be interacting with specific alpha subunit amino acids residues highlighting the direction of future experiments.

Genomic Properties and Temporal Analysis of the Interaction of an Invasive Escherichia albertii With Epithelial Cells

Diarrhea is one of the main causes of infant mortality worldwide, mainly in the developing world. Among the various etiologic agents, Escherichia albertii is emerging as an important human enteropathogen. E. albertii promote attaching and effacing (AE) lesions due to the presence of the locus of enterocyte effacement (LEE) that encodes a type three secretion system (T3SS), the afimbrial adhesin intimin and its translocated receptor, Tir, and several effector proteins. We previously showed that E. albertii strain 1551-2 invades several epithelial cell lineages by a process that is dependent on the intimin-Tir interaction. To understand the contribution of T3SS-dependent effectors present in E. albertii 1551-2 during the invasion process, we performed a genetic analysis of the LEE and non-LEE genes and evaluated the expression of the LEE operons in various stages of bacterial interaction with differentiated intestinal Caco-2 cells. The kinetics of the ability of the 1551-2 strain to colonize and form AE lesions was also investigated in epithelial HeLa cells. We showed that the LEE expression was constant during the early stages of infection but increased at least 4-fold during bacterial persistence in the intracellular compartment. An in silico analysis indicated the presence of a new tccP/espFU subtype, named tccP3. We found that the encoded protein colocalizes with Tir and polymerized F-actin during the infection process in vitro. Moreover, assays performed with Nck null cells demonstrated that the 1551-2 strain can trigger F-actin polymerization in an Nck-independent pathway, despite the fact that TccP3 is not required for this phenotype. Our study highlights the importance of the T3SS during the invasion process and for the maintenance of E. albertii 1551-2 inside the cells. In addition, this work may help to elucidate the versatility of the T3SS for AE pathogens, which are usually considered extracellular and rarely reach the intracellular environment.

Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7

ABSTRACT The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo. This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections. IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

Whole-Genome Analysis of a Shiga Toxin-Producing Escherichia coli O103:H2 Strain Isolated from Cattle Feces

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) serotype O103 is one of the primary pathogenic contaminants of beef products, contributing to several foodborne outbreaks in recent years. Here, we report the whole-genome sequence of a STEC O103:H2 strain isolated from cattle feces that contains a locus of enterocyte effacement (LEE) pathogenicity island.

Molecular Study of Regulatory Gene (Ler) in Enteropathogenic Escherichia Coli (EPEC) of Diarrheagenic Patients

The locus of enterocyte effacement LEE-encoded regulator (Ler( is a global regulator of multiple virulence genes expression in the Enteropathogenic Escherichia coli (EPEC), including those encoding the type III secretion pathway and adhesion proteins such as intimin. Ler is central to the process of the formation of the attaching and effacing (AE) lesions. This study aimed to perform the molecular detection of Ler gene in EPEC, since there is no related previous study in Iraq. Two hundred and fifty stool specimens from children under two years of age for both sexes were collected from some Iraqi hospitals. All isolates were diagnosed according to morphological characteristics and biochemical tests. The results showed that 140 (56%) samples were identified as E.coli, while 8 (5.7%) isolates were identified as EPEC as confirmed by using VITEK 2 system. Susceptibility test was determined for all EPEC isolates to 16 different antibiotics. The results showed that 100%of these isolates were resistant to Ampicillin, Cefazolin, Ceftriaxone, Cefepime, Trimethoprim and Ceftazidime, whereas resistance values to Nitrofurantoin, Cefoxitin and Gentamicin were 66%, 40%, and 15% respectively. However 100% of the isolates were sensitive to Piperacillin, Ertapenem, Imipenem, Amikacin, Ciprofloxacin, Levofloxacin and Tigecycline. Monoplex pattern of PCR was used for detecting 16SrRNA, eae, stx1, lifA and Ler genes in EPEC. The results showed that the isolates of E.coli were positive for 16SrRNA, eae, lifA and Ler, while no bands of stx1 appeared.

The Ethanolamine-Sensing Transcription Factor EutR Promotes Virulence and Transmission during Citrobacter rodentium Intestinal Infection

ABSTRACT Enteric pathogens exploit chemical and nutrient signaling to gauge their location within a host and control expression of traits important for infection. Ethanolamine-containing molecules are essential in host physiology and play important roles in intestinal processes. The transcription factor EutR is conserved in the Enterobacteriaceae and is required for ethanolamine sensing and metabolism. In enterohemorrhagic Escherichia coli (EHEC) O157:H7, EutR responds to ethanolamine to activate expression of traits required for host colonization and disease; however, the importance of EutR to EHEC intestinal infection has not been examined. Because EHEC does not naturally colonize or cause disease in mice, we employed the natural murine pathogen Citrobacter rodentium as a model of EHEC virulence to investigate the importance of EutR in vivo. EHEC and C. rodentium possess the locus of enterocyte effacement (LEE), which is the canonical virulence trait of attaching and effacing pathogens. Our findings demonstrate that ethanolamine sensing and EutR-dependent regulation of the LEE are conserved in C. rodentium. Moreover, during infection, EutR is required for maximal LEE expression, colonization, and transmission efficiency. These findings reveal that EutR not only is important for persistence during the primary host infection cycle but also is required for maintenance in a host population.

PchE Regulation of Escherichia coli O157:H7 Flagella, Controlling the Transition to Host Cell Attachment

Shiga toxins and intimate adhesion controlled by the locus of enterocyte effacement are major enterohemorrhagic Escherichia coli (EHEC) virulence factors. Curli fimbriae also contribute to cell adhesion and are essential biofilm components. The transcriptional regulator PchE represses the expression of curli and their adhesion to HEp-2 cells. Past studies indicate that pchE also represses additional adhesins that contribute to HEp-2 cell attachment. In this study, we tested for pchE regulation of several tissue adhesins and their regulators. Three adhesin-encoding genes (eae, lpfA1, fliC) and four master regulators (csgD, stpA, ler, flhDC) were controlled by pchE. pchE over-expression strongly up-regulated fliC but the marked flagella induction reduced the attachment of O157:H7 clinical isolate PA20 to HEp-2 cells, indicating that flagella were blocking cell attachments rather than functioning as an adhesin. Chemotaxis, motor, structural, and regulatory genes in the flagellar operons were all increased by pchE expression, as was PA20 motility. This study identifies new members in the pchE regulon and shows that pchE stimulates flagellar motility while repressing cell adhesion, likely to support EHEC movement to the intestinal surface early in infection. However, induced or inappropriate pchE-dependent flagellar expression could block cell attachments later during disease progression.