Locus Of Enterocyte Effacement Research Articles

Tight Junction Disruption Induced by Type 3 Secretion System Effectors Injected by Enteropathogenic and Enterohemorrhagic Escherichia coli.

The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption.

Development of an HTS system to identify natural chemicals that specifically inhibit Escherichia coli O157:H7 adhesion to host cells

Adhesion to intestinal epithelium is critical for virulence and infection of Escherichia coli O157:H7, which is a major cause of foodborne illnesses. Targeting adhesion but not growth of the pathogen is an effective approach to control bacterial infections. In this study, a high-throughput screening system was developed to identify anti-adhesive natural chemicals using a reporter strain whose activity entirely depends on Ler since genes encoding the locus of enterocyte effacement (LEE) which forms attaching and effacing lesions are known to be activated by Ler. A selected hit, yomogin, suppressed the expression of virulent genes encoded within the LEE without affecting bacterial growth itself. Yomogin significantly reduced the adherence of E. coli O157:H7 to Caco-2 epithelial cells and cytotoxicity. These results indicated that the developed system allows for rapid and cost-effective discovery of novel anti-adhesive agents and yomogin can prevent early stage of E. coli O157:H7 pathogenesis.

A secretome view of colonisation factors in Shiga toxin-encoding Escherichia coli (STEC): from enterohaemorrhagic E.coli (EHEC) to related enteropathotypes.

Shiga toxin-encoding Escherichia coli (STEC) regroup strains that carry genes encoding Shiga toxin (Stx). Among intestinal pathogenic E.coli, enterohaemorrhagic E.coli (EHEC) constitute the major subgroup of virulent STEC. EHEC cause serious human disease such as haemorrhagic colitis and haemolytic-uremic syndrome. While EHEC have evolved from enteropathogenic E.coli, hybrids with enteroaggregative E.coli have recently emerged. Of note, some enteroinvasive E.coli also belong to the STEC group. While the LEE (locus of enterocyte effacement) is a key and prominent molecular determinant in the pathogenicity, neither all EHEC nor STEC contain the LEE, suggesting that they possess additional virulence and colonisation factors. Currently, nine protein secretion systems have been described in diderm-lipopolysaccharide bacteria (archetypal Gram-negative) and can be involved in the secretion of extracellular effectors, cell-surface proteins or assembly of cell-surface organelles, such as flagella or pili. In this review, we focus on the secretome of STEC and related enteropathotypes, which are relevant to the colonisation of biotic and abiotic surfaces. Considering the wealth of potential protein trafficking mechanisms, the different combinations of colonisation factors and modulation of their expression is further emphasised with regard to the ecophysiology of STEC.

The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes bloody diarrhea and hemolytic uremic syndrome throughout the world. A defining feature of EHEC pathogenesis is the formation of attaching and effacing (AE) lesions on colonic epithelial cells. Most of the genes that code for AE lesion formation, including a type three secretion system (T3SS) and effectors, are carried within a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). In this study, we report that a putative regulator, which is encoded in the cryptic E. coli type three secretion system 2 (ETT2) locus and herein renamed EtrB, plays an important role in EHEC pathogenesis. The etrB gene is expressed as a monocistronic transcript, and EtrB autoregulates expression. We provide evidence that EtrB directly interacts with the ler regulatory region to activate LEE expression and promote AE lesion formation. Additionally, we mapped the EtrB regulatory circuit in EHEC to determine a global role for EtrB. EtrB is regulated by the transcription factor QseA, suggesting that these proteins comprise a regulatory circuit important for EHEC colonization of the gastrointestinal tract.

Bacterial Adrenergic Sensors Regulate Virulence of Enteric Pathogens in the Gut.

ABSTRACTEnteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium. Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine β-hydroxylase knockout (Dbh−/−) mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a noninvasive pathogen.

Pathogenicity Island O-122 in enteropathogenic Escherichia coli strains is associated with diarrhea severity in children from Lima Peru

EPEC is an attaching and effacing diarrheal pathogen that carries a large pathogenicity island, locus for enterocyte effacement (LEE). Recently, the pathogenicity island PAI O-122 was described among non-LEE effectors and found to be associated with diarrhea among atypical EPEC strains. It is unknown if incomplete PAI O-122 could be associated with diarrhea duration and severity. To identify these virulence determinants we analyzed 379 EPEC strains isolated from Peruvian children. EPEC was diagnosed by PCR(eae+, stx-) and classified as typical(t-EPEC) or atypical(a-EPEC). To characterize PAI O-122 we amplified three modules by PCR: Module 1(pagC), Module 2(senA, nleB and nleE) and Module 3(lifA/efa-1). To characterize the large ORF lifA/efa-1 we amplified the regions known as efa-N, efa-M and efa-C. Clinical information was obtained from the cohort study. A total of 379 EPEC strains were able to analyze PAI O-122 genes, 128 (10.4%) EPEC strains were isolated from 1235 diarrhea episodes and 251(9.2%) from 2734 healthy controls. t-EPEC strains were isolated from 14.8% (19/128) of children with diarrhea and 25/251(10.0%) from healthy controls. The most frequent PAI O-122 genes were nleE(37.7%), senA(34.6%) and nleB(37.5%), with similar prevalence among diarrhea and control samples. However, lifA/efa-1 was more common among diarrhea cases than healthy control cases (30.5% vs. 21.1%, p<0.05). The presence of complete PAI O-122 was associated with diarrhea episodes of higher severity among single pathogen infection (33.3% vs. 1.8%, p<0.05) mainly due to the presence of a complete lifA/efa-1 gene. In summary, the gene lifA/efa-1 is significantly associated with diarrheal episodes of higher severity, suggesting to be an important virulent factor.

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments.

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx1a eae-β1 strain was associated with a large number of virulence-associated genes (n = 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx1a, stx2a, stx2c, and stx2d) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2. The species E. coli includes a wide variety of strains, some of which may be responsible for severe infections. This study, a molecular risk assessment study of E. coli strains isolated from the coastal environment, was conducted to evaluate the potential risk for shellfish consumers. This report describes the characterization of virulence gene profiles and stx/eae polymorphisms of E. coli isolates and clearly highlights the finding that the majority of strains isolated from coastal environment are potentially weakly pathogenic, while some are likely to be more pathogenic.

Virulence Gene Profiles and Clonal Relationships of Escherichia coli O26:H11 Isolates from Feedlot Cattle as Determined by Whole-Genome Sequencing

Escherichia coli O26 is the second most important enterohemorrhagic E. coli (EHEC) serogroup worldwide. Serogroup O26 strains are categorized mainly into two groups: enteropathogenic (EPEC) O26, carrying a locus of enterocyte effacement (LEE) and mostly causing mild diarrhea, and Shiga-toxigenic (STEC) O26, which carries the Shiga toxin (STX) gene (stx), responsible for more severe outcomes. stx-negative O26 strains can be further split into two groups. One O26 group differs significantly from O26 EHEC, while the other O26 EHEC-like group shows all the characteristics of EHEC O26 except production of STX. In order to determine the different populations of O26 E. coli present in U.S. cattle, we sequenced 42 O26:H11 strains isolated from feedlot cattle and compared them to 37 O26:H11 genomes available in GenBank. Phylogenetic analysis by whole-genome multilocus sequence typing (wgMLST) showed that O26:H11/H(-) strains in U.S. cattle were highly diverse. Most strains were sequence type 29 (ST29). By wgMLST, two clear lineages could be distinguished among cattle strains. Lineage 1 consisted of O26:H11 EHEC-like strains (ST29) (4 strains) and O26:H11 EHEC strains (ST21) (2 strains), and lineage 2 (36 strains) consisted of O26:H11 EPEC strains (ST29). Overall, our analysis showed U.S. cattle carried pathogenic (ST21; stx1 (+) ehxA(+) toxB(+)) and also potentially pathogenic (ST29; ehxA(+) toxB(+)) O26:H11 E. coli strains. Furthermore, in silico analysis showed that 70% of the cattle strains carried at least one antimicrobial resistance gene. Our results showed that whole-genome sequence analysis is a robust and valid approach to identify and genetically characterize E. coli O26:H11, which is of importance for food safety and public health. Escherichia coli O26 is the second most important type of enterohemorrhagic E. coli (EHEC) worldwide. Serogroup O26 strains are categorized into two groups: enteropathogenic (EPEC) carrying LEE, causing mild diarrhea, and Shiga toxigenic (STEC) carrying the stx gene, responsible for more severe outcomes. However, there are currently problems in distinguishing one group from the other. Furthermore, several O26 stx-negative strains are consistently misidentified as either EHEC-like or EPEC. The use of whole-genome sequence (WGS) analysis of O26 strains from cattle in the United States (i) allowed identification of O26 strains present in U.S. cattle, (ii) determined O26 strain diversity, (iii) solved the misidentification problem, and (iv) screened for the presence of antimicrobial resistance and virulence genes in the strains. This study provided a framework showing how to easily and rapidly use WGS information to identify and genetically characterize E. coli O26:H11, which is important for food safety and public health.

Development of a High Resolution Virulence Allelic Profiling (HReVAP) Approach Based on the Accessory Genome of Escherichia coli to Characterize Shiga-Toxin Producing E. coli (STEC).

Shiga-toxin producing Escherichia coli (STEC) strains possess a large accessory genome composed of virulence genes existing in multiple allelic variants, which sometimes segregate with specific STEC subpopulations. We analyzed the allelic variability of 91 virulence genes of STEC by Real Time PCR followed by melting curves analysis in 713 E. coli strains including 358 STEC. The 91 genes investigated were located on the locus of enterocyte effacement (LEE), OI-57, and OI-122 pathogenicity islands and displayed a total of 476 alleles in the study population. The combinations of the 91 alleles of each strain were termed allelic signatures and used to perform cluster analyses. We termed such an approach High Resolution Virulence Allelic Profiling (HReVAP) and used it to investigate the phylogeny of STEC of multiple serogroups. The dendrograms obtained identified groups of STEC segregating approximately with the serogroups and allowed the identification of subpopulations within the single groups. The study of the allelic signatures provided further evidence of the coevolution of the LEE and OI-122, reflecting the occurrence of their acquisition through a single event. The HReVAP analysis represents a sensitive tool for studying the evolution of LEE-positive STEC.

Subtilase cytotoxin produced by locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli induces stress granule formation.

Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)-negative strains of Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase-dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB-induced SG formation is regulated by activation of double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH4 Cl and chloroquine, suppressed SubAB-induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death-associated protein 1 (DAP1) knockdown increased basal phospho-PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA-treated cells. Our findings show that SubAB-induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin-induced SG formation.

The Two-Component System CpxRA Negatively Regulates the Locus of Enterocyte Effacement of Enterohemorrhagic Escherichia coli Involving σ(32) and Lon protease.

Enterohemorrhagic Escherichia coli (EHEC) is a significant cause of serious human gastrointestinal disease worldwide. EHEC strains contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes virulence factors responsible for damaging the gut mucosa. The Cpx envelope stress response of E. coli is controlled by a two-component system (TCS) consisting of a sensor histidine kinase (CpxA) and a cytoplasmic response regulator (CpxR). In this study, we investigated the role of CpxRA in the expression of LEE-encoded virulence factors of EHEC. We found that a mutation in cpxA significantly affected adherence of EHEC to human epithelial cells. Analysis of this mutant revealed the presence of high levels of CpxR which repressed transcription of grlA and ler, the main positive virulence regulators of the LEE, and influenced negatively the production of the type 3 secretion system–associated EspABD translocator proteins. It is known that CpxR activates rpoH (Sigma factor 32), which in turns activates transcription of the lon protease gene. We found that transcription levels of ler and grlA were significantly increased in the lon and cpxA lon mutants suggesting that lon is involved in down-regulating LEE genes. In addition, the Galleria mellonella model of infection was used to analyze the effect of the loss of the cpx and lon genes in EHEC's ability to kill the larvae. We found that the cpxA mutant was significantly deficient at killing the larvae however, the cpxA lon mutant which overexpresses LEE genes in vitro, was unable to kill the larvae, suggesting that virulence in the G. mellonella model is T3SS independent and that CpxA modulates virulence through a yet unknown EHEC-specific factor. Our data provides new insights and broadens our scope into the complex regulatory network of the LEE in which the CpxA sensor kinase plays an important role in a cascade involving both global and virulence regulators.

Evolution of atypical enteropathogenic E. coli by repeated acquisition of LEE pathogenicity island variants.

Atypical enteropathogenic Escherichia coli (aEPEC) is an umbrella term given to E. coli that possess a type III secretion system encoded in the locus of enterocyte effacement (LEE), but lack the virulence factors (stx, bfpA) that characterize enterohaemorrhagic E. coli and typical EPEC, respectively. The burden of disease caused by aEPEC has recently increased in industrialized and developing nations, yet the population structure and virulence profile of this emerging pathogen are poorly understood. Here, we generated whole-genome sequences of 185 aEPEC isolates collected during the Global Enteric Multicenter Study from seven study sites in Asia and Africa, and compared them with publicly available E. coli genomes. Phylogenomic analysis revealed ten distinct widely distributed aEPEC clones. Analysis of genetic variation in the LEE pathogenicity island identified 30 distinct LEE subtypes divided into three major lineages. Each LEE lineage demonstrated a preferred chromosomal insertion site and different complements of non-LEE encoded effector genes, indicating distinct patterns of evolution of these lineages. This study provides the first detailed genomic framework for aEPEC in the context of the EPEC pathotype and will facilitate further studies into the epidemiology and pathogenicity of EPEC by enabling the detection and tracking of specific clones and LEE variants.

Mechanosensing regulates virulence in Escherichia coli O157:H7

ABSTRACTEnterohemorrhagic Escherichia coli O157:H7 is a food-borne pathogen transmitted via the fecal-oral route, and can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in the human host. Although a range of colonization factors, Shiga toxins and a type III secretion system (T3SS) all contribute to disease development, the locus of enterocyte effacement (LEE) encoded T3SS is responsible for the formation of lesions in the intestinal tract. While a variety of chemical cues in the host environment are known to up-regulate LEE expression, we recently demonstrated that changes in physical forces at the site of attachment are required for localized, full induction of the system and thus spatial regulation of virulence in the intestinal tract. Here, we discuss our findings in the light of other recent studies describing mechanosensing of the host and force-dependent induction of virulence mechanisms. We discuss potential mechanisms of mechanosensing and mechanotransduction, and the level of conservation across bacterial species.

Interkingdom Chemical Signaling in Enterohemorrhagic Escherichia coli O157:H7.

Escherichia coli is one of the most-studied species of bacteria due to its frequent incidence in diverse environments and hosts, as well as its use as a tool in molecular biology. Most E. coli strains are commensal, in that they colonize the host without causing disease; however, some strains of E. coli are pathogens and are able to cause diverse illnesses, including urinary tract infections, sepsis/meningitis, as well as intestinal disease that result in diarrhea (Kaper et al. 2004). Six categories of diarrheagenic E. coli are recognized, and these are classified in part based on how they interact with epithelial cells (Kaper et al. 2004). Of these, enterohemorrhagic E. coli O157:H7 (EHEC) is one of the most important pathogenic E. coli strains. EHEC causes major outbreaks of bloody diarrhea that can result in the development of fatal hemorrhagic colitis and hemolytic uremic syndrome (Karmali et al. 1983). EHEC colonizes the colon, where it forms attaching and effacing (AE) lesions on the intestinal epithelial cell. AE lesions are characterized by intimate attachment of EHEC to epithelial cells, effacement of the microvilli and rearrangement of the underlying cytoskeleton, which results in formation of a pedestal-like structure beneath the bacterium (Jerse et al. 1990; Jarvis et al. 1995; Kenny et al. 1997). Most of the genes involved in the formation of AE lesions are encoded within a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE) (McDaniel et al. 1995). The LEE contains 41 genes that are organized in five major operons (LEE1, LEE2, LEE3, LEE5, and LEE4) (Elliott et al. 1998, 1999; Mellies et al. 1999). The LEE encodes a type three secretion system (T3SS) (Jarvis et al. 1995), an adhesin (intimin) (Jerse et al. 1990) and its receptor (Tir) (Kenny et al. 1997), as well as effector proteins (Kenny et al. 1996; Abe et al. 1997; McNamara and Donnenberg 1998; Elliott et al. 2001; Tu et al. 2003; Kanack et al. 2005). EHEC also encodes an arsenal of effector proteins located outside of the LEE that are important in EHEC virulence (Campellone et al. 2004; Deng et al. 2004; Garmendia et al. 2004, 2005; Gruenheid et al. 2004; Tobe et al. 2006).

The Surface Sensor NlpE of Enterohemorrhagic Escherichia coli Contributes to Regulation of the Type III Secretion System and Flagella by the Cpx Response to Adhesion.

Although the adhesion of enterohemorrhagic Escherichia coli (EHEC) is central to the EHEC-host interaction during infection, it remains unclear how such adhesion regulates virulence factors. Adhesion to abiotic surfaces by E. coli has been reported to be an outer membrane lipoprotein NlpE-dependent activation cue of the Cpx pathway. Therefore, we investigated the role of NlpE in EHEC on the adhesion-mediated expression of virulence genes. NlpE in EHEC contributed to upregulation of the locus of enterocyte effacement (LEE) genes encoded type III secretion system and to downregulated expression of the flagellin gene by activation of the Cpx pathway during adherence to hydrophobic glass beads and undifferentiated Caco-2 cells. Moreover, LysR homologue A (LrhA) in EHEC was involved in regulating the expression of the LEE genes and flagellin gene in response to adhesion. Gel mobility shift analysis revealed that response regulator CpxR bound to the lrhA promoter region and thereby regulated expressions of the LEE genes and flagellin gene via the transcriptional regulator LrhA in EHEC. Therefore, these results suggest that the sensing of adhesion signals via NlpE is important for regulation of the expression of the type III secretion system and flagella in EHEC during infection.

Defining the Genome Features of Escherichia albertii, an Emerging Enteropathogen Closely Related to Escherichia coli.

Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified. Here, we sequenced the genome of 29 E. albertii strains (3 complete and 26 draft sequences) isolated from multiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1 Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors and a possibly active second T3SS known as ETT2 (E. coli T3SS 2) that is inactivated in E. coli. Although this organism has been observed to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based on these results, we have developed a nested polymerase chain reaction system to directly detect E. albertii. Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen.

Global Regulator of Virulence A (GrvA) Coordinates Expression of Discrete Pathogenic Mechanisms in Enterohemorrhagic Escherichia coli through Interactions with GadW-GadE.

Global regulator of virulence A (GrvA) is a ToxR-family transcriptional regulator that activates locus of enterocyte effacement (LEE)-dependent adherence in enterohemorrhagic Escherichia coli (EHEC). LEE activation by GrvA requires the Rcs phosphorelay response regulator RcsB and is sensitive to physiologically relevant concentrations of bicarbonate, a known stimulant of virulence systems in intestinal pathogens. This study determines the genomic scale of GrvA-dependent regulation and uncovers details of the molecular mechanism underlying GrvA-dependent regulation of pathogenic mechanisms in EHEC. In a grvA-null background of EHEC strain TW14359, RNA sequencing analysis revealed the altered expression of over 700 genes, including the downregulation of LEE- and non-LEE-encoded effectors and the upregulation of genes for glutamate-dependent acid resistance (GDAR). Upregulation of GDAR genes corresponded with a marked increase in acid resistance. GrvA-dependent regulation of GDAR and the LEE required gadE, the central activator of GDAR genes and a direct repressor of the LEE. Control of gadE by GrvA was further determined to occur through downregulation of the gadE activator GadW. This interaction of GrvA with GadW-GadE represses the acid resistance phenotype, while it concomitantly activates the LEE-dependent adherence and secretion of immune subversion effectors. The results of this study significantly broaden the scope of GrvA-dependent regulation and its role in EHEC pathogenesis. Enterohemorrhagic Escherichia coli (EHEC) is an intestinal human pathogen causing acute hemorrhagic colitis and life-threatening hemolytic-uremic syndrome. For successful transmission and gut colonization, EHEC relies on the glutamate-dependent acid resistance (GDAR) system and a type III secretion apparatus, encoded on the LEE pathogenicity island. This study investigates the mechanism whereby the DNA-binding regulator GrvA coordinates activation of the LEE with repression of GDAR. Investigating how these systems are regulated leads to an understanding of pathogenic behavior and novel strategies aimed at disease prevention and control.

Genomic insights into the TTSS island of enteropathogenic E. coli and Salmonella and its conjugational transfer

Whole genome sequences of the non-pathogenic K12 and pathogenic O127:H6 strains of Escherichia coli were analyzed using Mauve software. The genomes showed 80% similarity with few desert regions including a 35.99 kb region in pathogenic strain. This region contained Locus of Enterocyte Effacement (LEE) and Type III Secretion System (TTSS) genes. Whole genome alignment of this E. coli pathogenic strain and Salmonella enterica subsp. enterica serovar Newport str. SK254 showed 40% homology. Intimin protein coding escU gene of E. coli and ssaU gene of S. enterica were analyzed by BLASTn and ClustalW and cross referred with Pathogenicity Island Database (PDI DB) to check similarity with other foodborne bacterial species. The ssaU gene of Salmonella was found to be designated as escU in E. coli database. Comparison of these genes at nucleotide and amino acid sequence level revealed possible codon redundancies. Six clinical isolates of E. coli (designated as SBANU 1 to 6) were screened for salt aggregation and hemolysin production abilities followed by PCR analysis of escU gene. The E. coli isolate SBANU 6 was further used in induced conjugation assay with S. enterica as donor. PCR analysis and sequencing of the amplified DNA in E. coli transconjugant cells revealed the possible acquiring of ssaU gene from S. enterica.

Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages, Providing New Insights into the Evolution of EHEC.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.

Comparative Genomics Provides Insight into the Diversity of the Attaching and Effacing Escherichia coli Virulence Plasmids.

Attaching and effacing Escherichia coli (AEEC) strains are a genomically diverse group of diarrheagenic E. coli strains that are characterized by the presence of the locus of enterocyte effacement (LEE) genomic island, which encodes a type III secretion system that is essential to virulence. AEEC strains can be further classified as either enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC), or atypical EPEC, depending on the presence or absence of the Shiga toxin genes or bundle-forming pilus (BFP) genes. Recent AEEC genomic studies have focused on the diversity of the core genome, and less is known regarding the genetic diversity and relatedness of AEEC plasmids. Comparative genomic analyses in this study demonstrated genetic similarity among AEEC plasmid genes involved in plasmid replication conjugative transfer and maintenance, while the remainder of the plasmids had sequence variability. Investigation of the EPEC adherence factor (EAF) plasmids, which carry the BFP genes, demonstrated significant plasmid diversity even among isolates within the same phylogenomic lineage, suggesting that these EAF-like plasmids have undergone genetic modifications or have been lost and acquired multiple times. Global transcriptional analyses of the EPEC prototype isolate E2348/69 and two EAF plasmid mutants of this isolate demonstrated that the plasmid genes influence the expression of a number of chromosomal genes in addition to the LEE. This suggests that the genetic diversity of the EAF plasmids could contribute to differences in the global virulence regulons of EPEC isolates.