Locus Of Enterocyte Effacement Research Articles

The Enteropathogenic E. coli (EPEC) Tir Effector Inhibits NF-κB Activity by Targeting TNFα Receptor-Associated Factors

Enteropathogenic Escherichia coli (EPEC) disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE) Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle) effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir) effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF) adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function.

Genetic Characterization of Escherichia coli O157:H7 Strains Isolated from the One-Humped Camel (Camelus dromedarius) by Using Microarray DNA Technology

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.

Low prevalence of STEC autotransporter contributing to biofilm formation (Sab) in verocytotoxin-producing Escherichia coli isolates of humans and raw meats

Escherichia coli autotransporters (AT) are known to confer adherence to eukaryotic extracellular matrix and may, therefore, be virulence-associated. Recently, the plasmid-borne STEC AT contributing to biofilm formation (Sab) was described in verocytotoxin (VT)-producing E. coli (VTEC) strains that do not carry the locus of enterocyte effacement (LEE). Using polymerase chain reaction (PCR), we investigated the prevalence of sab and other virulence genes, VT1 (vtx1), VT2 (vtx2), intimin (eae), enterohemolysin (ehxA), STEC autoagglutinating adhesin (saa), and subtilase cytotoxin (subA), in VTEC isolates from patients (n=263) and raw meats of ruminants and wildlife (n=104) in Belgium from 1990 to 2010. Overall, sab was detected in three (0.82%) of 367 VTEC strains comprising human isolates of serotypes O162:H28 (no clinical data available) and OX183:H18 (patient with abdominal pain), and one ground beef O181:H16 isolate. These three sab-positive isolates were eae-negative, but ehxA-, saa-, and subA-positive. Our data show that sab is uncommon in VTEC isolates. All sab-positive VTEC strains identified to date carried a comparable plasmid-bound virulence profile (ehxA-saa-subA-sab), which may be transmitted to other strains. Sab may mediate intestinal adherence in some LEE-negative VTEC isolates, but more studies on its prevalence and function are needed.

Hfq Virulence Regulation in Enterohemorrhagic Escherichia coli O157:H7 Strain 86-24

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.

Direct and indirect control of Lrp on LEE pathogenicity genes of Citrobacter rodentium

Citrobacter rodentium is a mouse pathogen that, because of its similarities with human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) strains of Escherichia coli is widely used as a model system for in vivo and in vitro studies. Similarly to EPEC and EHEC, C. rodentium carries the LEE (locus of enterocyte effacement) pathogenicity island, encoding virulence factors essential for causing transmissible colonic hyperplasia in mice by attaching and effacing (A/E) lesions. Expression of the genes carried by the LEE pathogenicity island is controlled by complex networks of transcriptional factors, including the global regulators H-NS, IHF, and Fis. In this study, we analyzed the role of Lrp, another global regulator of gene expression in enteric bacteria, on the expression of LEE genes of C. rodentium. To this aim, a real-time PCR approach was used and revealed a negative role of Lrp on the expression of all analyzed LEE genes. Mobility-shift experiments indicated that Lrp action is direct on LEE1 and indirect on all other analyzed LEE genes.

Enteropathogenic Escherichia coli infection in children

Enteropathogenic Escherichia coli (EPEC) is an important diarrheal pathogen of young children. As the diagnosis of EPEC is now based mainly on molecular criteria, there has been an important change in its prevalence. The purpose of this study is to review the current epidemiology of EPEC infection and the new insights into its physiopathology. Recent epidemiological studies indicate that atypical EPEC (aEPEC) is more prevalent than typical EPEC (tEPEC) in both developed and developing countries, and that aEPEC is important in both pediatric endemic diarrhea and diarrhea outbreaks. Therefore, it is important to further characterize the pathogenicity of these emerging strains. The virulence mechanisms and physiopathology of the attaching and effacing lesion (A/E) and the type three secretion-system (T3SS) are complex but well studied. A/E strains use their pool of locus of enterocyte effacement (LEE)-encoded and non-LEE-encoded effector proteins to subvert and modulate cellular and barrier properties of the host. However, the exact mechanisms of diarrhea in EPEC infection are not completely understood. Remarkable progress has been made to identify virulence determinants required to mediate the pathogenesis of EPEC. However, fast, easy, and inexpensive diagnostic methods are needed in order to define optimal treatment and prevention for children in endemic areas.

PerC and GrlA independently regulate Ler expression in enteropathogenic Escherichia coli

Ler, encoded by the locus of enterocyte effacement (LEE) of attaching and effacing (A/E) pathogens, induces the expression of LEE genes by counteracting the silencing exerted by H-NS. Ler expression is modulated by several global regulators, and is activated by GrlA, which is also LEE-encoded. Typical enteropathogenic Escherichia coli (EPEC) strains contain the EAF plasmid, which carries the perABC locus encoding PerC. The precise role of PerC in EPEC virulence gene regulation has remained unclear, mainly because EPEC strains lacking the pEAF still express the LEE genes and because PerC is not present in other A/E pathogens such as Citrobacter rodentium. Here, we describe that either PerC or GrlA can independently activate ler expression and, in consequence, of LEE genes depending on the growth conditions. Both PerC and GrlA, with the aid of IHF, counteract the repression exerted by H-NS on ler and can also further increase its activity. Our results substantiate the role of PerC and GrlA in EPEC virulence gene regulation and suggest that these convergent regulatory mechanisms may have represented an evolutionary adaptation in EPEC to co-ordinate the expression of plasmid- and chromosome-encoded virulence factors needed to successfully colonize its intestinal niche.

Dual regulatory pathways of flagellar gene expression by ClpXP protease in enterohaemorrhagic Escherichia coli

In enterobacteria such as Escherichia coli and Salmonella species, flagellar biogenesis is strictly dependent upon the master regulator flhDC. Here, we demonstrate that in enterohaemorrhagic E. coli (EHEC), the flagellar regulon is controlled by ClpXP, a member of the ATP-dependent protease family, through two pathways: (i) post-translational control of the FlhD/FlhC master regulator and (ii) transcriptional control of the flhDC operon. Both FlhD and FlhC proteins accumulated markedly following ClpXP depletion, and their half-lives were significantly longer in the mutant cells, suggesting that ClpXP is responsible for degrading FlhD and FlhC proteins, leading to downregulation of flagellar expression. ClpXP was involved in regulating the transcription of the flhD promoter only when the cells had entered stationary phase in a culture medium that markedly induced expression of the locus of enterocyte effacement (LEE). Comparative analyses of transcription from the flhD promoter in EHEC cells with different genetic backgrounds suggested that the downregulation of flhDC expression by ClpXP is dependent on the LEE-encoded GrlR-GrlA system. We have also shown that the degradation of FlhD and FlhC by ClpXP is responsible for downregulating flagellar expression even when LEE expression is induced.

Coordinate Control of the Locus of Enterocyte Effacement and Enterohemolysin Genes by Multiple Common Virulence Regulators in Enterohemorrhagic Escherichia coli

The locus of enterocyte effacement (LEE) pathogenicity island is required for the intimate adhesion of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelial cells. GrlR and GrlA are LEE-encoded negative and positive regulators, respectively. The interaction of these two regulators is important for controlling the transcription of LEE genes through Ler, a LEE-encoded central activator for the LEE. The GrlR-GrlA regulatory system controls not only LEE but also the expression of the flagellar and enterohemolysin (Ehx) genes in EHEC. Since Ehx levels were markedly induced in a grlR mutant but not in a grlR grlA double mutant and significantly increased by overexpression of GrlA in a ler mutant, GrlA is responsible for this regulation (T. Saitoh et al., J. Bacteriol. 190:4822-4830, 2008). In this study, additional investigations of the regulation of ehx gene expression determined that Ler also acts as an activator for Ehx expression without requiring GrlA function. We recently reported that the LysR-type regulator LrhA positively controls LEE expression (N. Honda et al., Mol. Microbiol. 74:1393-1411, 2009). The hemolytic activity of the lrhA mutant strain of EHEC was lower than that of the wild-type strain, and LrhA markedly induced ehx transcription in an E. coli K-12 strain, suggesting that LrhA also activates the transcription of ehx without GrlA and Ler. Gel mobility shift assays demonstrated that Ler and LrhA directly bind to the regulatory region of ehxC. Together, these results indicate that transcription of ehx is positively regulated by Ler, GrlA, and LrhA, which all act as positive regulators for LEE expression.

Enteroaggregative E. coli O104 from an outbreak of HUS in Germany 2011, could it happen again?

Enterohaemorrhagic E. coli (EHEC) particularly O157:H7 (Sequence type 11 complex), is the best documented and most well-known of E. coli that cause diarrhoea. The importance of EHEC lies in the severity of disease. Outbreaks can infect thousands of people causing bloody diarrhoea and haemolytic uremic syndrome (HUS) that in turn can result in protracted illness or even death. The ability of EHEC to colonise the human gut is normally associated with the presence of genes from another group of diarrhoeagenic E. coli, the enteropathogenic E. coli (EPEC), via the locus of enterocyte effacement. However, the massive outbreak in Germany was caused by an EHEC which had acquired virulence genes from yet another group of diarrhoeagenic E. coli, the enteroaggregative E. coli (EAEC). In reality EAEC is probably the most common bacterial cause of diarrhoea but is not identified in most diagnostic laboratories. This outbreak emphasises the importance of being able to detect all diarrhoeagenic E. coli and not to focus on E. coli O157:H7 alone. Routine surveillance systems for EAEC, a once ignored global pathogen, would go a long way to reaching this goal. This review describes methods for identifying non-O157 EHEC and describes the key genetic features of EHEC and EAEC. Our aim is to provide information for laboratories and policy makers which enables them to make informed decisions about the best methods available for detecting newly emergent strains of diarrhoeagenic E. coli.

OL-031 In vivo investigation of Killed Leishmania Vaccine's (KLV) efficacy with Imiquimod (IMQ) as adjuvant in inhibition of visceralization of Leishmania major in Balb/c model

Background: Escherichia coli O157:H7 is associated with hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolyticuremic syndrome in humans. In addition to producing Shiga-like (Vero) toxin and enterohemolysin, E. coli O157:H7 has been shown to attach to the cytoplasmic membranes of intestinal epithelial cells, to efface their microvilli, and to cause actin to accumulate beneath sites of bacterial attachment. These features are shared with several other enterohemorrhagic E. coli (EHEC) serotypes and members of the enteropathogenic E. coli (EPEC) group. The eae gene, which has been shown to be necessary for attaching and effacing activity, encodes a 94to 97-kDa outer membrane protein (OMP) which is termed intimin. This gene is located in a chromosomal pathogenicity island also known as the locus of enterocyte effacement (LEE). The gold aim of this study was cloning of eae gene of E. coli O157:T in E. coli TOP10F strain. Methods: The eae gene was isolated from total genome of E. coli O157:T by PCR and eae specific PCR primers. DNA fragment of eae gene was cloned by T/A cloning technique in pGEMT easy vector (Invitrogen, San Diego, Calif.), and this construct transformed into E. coli TOP10F strain. Results: The results show that, eae was cloned in E. coli successfully. The sequencing result confirm that eae gene was cloned is correct and BLAST outcome demonstrated the sequences of eae is approved. Conclusions: Therefore it seems that the DNA constract that was produced in this study can be used for DNA vaccine against eae of E. coli O157:T in future researchs.

Adherence Pheno-genotypic of Escherichia coli O157:H7 Isolated from Beef, Feces of Cattle, Chicken and Human

Generally, adherence of micro-organisms to host cells is the frst step of the colonization to host surfaces. Escherichia coli O157:H7 can colonize to the intestine and induce attaching-effacing (AE) lessions. The capacity of inducing AE lesions is encoded by a pahtogenicity island, the locus of enterocyte effacement (LEE) that contains genes involved in generation of attaching and effaching (A/E) lesions. Among which that, the eae gene is encoding intimin, an outer membrane protein that is responsible to intimate attachment to the intestinal epithelial cells. A total of 20 local isolates obtained from human clinically, beef, cattle, chicken, and human non-clinically were tested to adherence pheno-genotypic of E. coli O157:H7. The eae gene was identifed using polymerase chain reaction with a specifc primer i.e AE19 forward and AE20 reverse. To confrm phenotypic of gene, further study was performed by culturing the bacteria in vero cell, followed by Giemsa staining and Acridine Orange Fluorescent staining 3 h and 6 h after incubation, respectivelly. Result of study showed that there were 19 out of 20 (95%) isolates identifed positive eae gene. Giemsa staining appeared that the bacteria with positive eae gene performed a cluster arroud of cell (localized adherence). On the other hand, the negative eae gene appeared as a diffuse adherence (DA). The study indicated that almost all of E. coli O157:H7 local isolates which was positive eae gene had potency to colonize to the intestine and induce attching-effaching lessions, and also cause cytopahatic effects in intestinal epithelial cell

Serological response of Shiga toxin-producing Escherichia coli type III secreted proteins in sera from vaccinated rabbits, naturally infected cattle, and humans.

Escherichia coli O157:H7 is an important zoonotic pathogen, causing hemolytic uremic syndrome (HUS). The colonization of cattle and human hosts is mediated through the action of effectors secreted via a type III secretion system (T3SS). The structural genes for the T3SS and many of the secreted effectors are located on a pathogenicity island called the locus of enterocyte effacement (LEE). We cloned and expressed the genes coding for 66 effectors and purified each to measure the cross-reactivity of type III secreted proteins from Shiga toxin-producing Escherichia coli (STEC) serotypes. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with type III secreted proteins (T3SPs) from four STEC serotypes, experimentally infected cattle, and human sera from six HUS patients. Twenty proteins were recognized by at least one of the STEC T3SP-vaccinated rabbits by Western blotting. Several structural proteins (EspA, EspB, and EspD) and a number of effectors (Tir, NleA, and TccP) were recognized by O26-, O103-, O111-, and O157-specific sera. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA, and NleA were recognized by the majority of the samples tested. A number of other proteins also were recognized by individual serum samples. Overall, proteins such as Tir, EspB, EspD, NleA, and EspA were highly immunogenic in vaccinated and naturally infected subjects and could be candidates for a cross-protective STEC vaccine.

Evaluation of hha and hha sepB mutant strains of Escherichia coli O157:H7 as bacterins for reducing E. coli O157:H7 shedding in cattle

Escherichia coli O157:H7 colonizes cattle intestines by using the locus of enterocyte effacement (LEE)-encoded proteins. The induction of systemic immune response against LEE-encoded proteins, therefore, will prove effective in reducing E. coli O157:H7 colonization in cattle. The previous studies have demonstrated that a hha (encodes for a hemolysin expression modulating protein) deletion enhances expression of LEE-encoded proteins and a sepB (encodes an ATPase required for the secretion of LEE-encoded proteins) deletion results in intracellular accumulation of LEE proteins. In this study, we demonstrate the efficacy of the hha and hha sepB deletion mutants as bacterins for reducing fecal shedding of E. coli O157:H7 in experimentally inoculated weaned calves. The weaned calves were injected intramuscularly with the bacterins containing 109 heat-killed cells of the hha+ wild-type or hha or hha sepB isogenic mutants, and boosted with the same doses 2- and 4-weeks later. The evaluation of the immune response two weeks after the last booster immunization revealed that the calves vaccinated with the hha mutant bacterin had higher antibody titers against LEE proteins compared to the titers for these antibodies in the calves vaccinated with the hha sepB mutant or hha+ wild-type bacterins. Following oral inoculations with 1010 CFU of the wild-type E. coli O157:H7, the greater numbers of calves in the group vaccinated with the hha or hha sepB mutant bacterins stopped shedding the inoculum strain within a few days after the inoculations compared to the group of calves vaccinated with the hha+ wild-type bacterin or PBS sham vaccine. Thus, the use of bacterins prepared from the hha and hha sepB mutants for reducing colonization of E. coli O157:H7 in cattle could represent a potentially important pre-harvest strategy to enhance post-harvest safety of bovine food products, water and produce.

Cross-reactive protection against enterohemorrhagic Escherichia coli infection by enteropathogenic E. coli in a mouse model.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related attaching and effacing (A/E) pathogens. The genes responsible for the A/E pathology are carried on a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Both pathogens share a high degree of homology in the LEE and additional O islands. EHEC prevalence is much lower in areas where EPEC is endemic. This may be due to the development of antibodies against common EPEC and EHEC antigens. This study investigated the hypothesis that EPEC infections may protect against EHEC infections. We used a mouse model to inoculate BALB/c mice intragastrically, first with EPEC and then with EHEC (E. coli O157:H7). Four control groups received either a nonpathogenic E. coli (NPEC) strain followed by EHEC (NPEC/EHEC), phosphate-buffered saline (PBS) followed by EHEC (PBS/EHEC), EPEC/PBS, or PBS/PBS. Mice were monitored for weight loss and symptoms. EPEC colonized the intestine after challenge, and mice developed serum antibodies to intimin and E. coli secreted protein B (encoded in the LEE). Prechallenge with an EPEC strain had a protective effect after EHEC infection, as only a few mice developed mild symptoms, from which they recovered. These mice had an increase in body weight similar to that in control animals, and tissue morphology exhibited mild intestinal changes and normal renal histology. All mice that were not prechallenged with the EPEC strain developed mild to severe symptoms after EHEC infection, with weight loss as well as intestinal and renal histopathological changes. These data suggest that EPEC may protect against EHEC infection in this mouse model.

Differential Gene Expression and Adherence of Escherichia coli O157:H7 In Vitro and in Ligated Pig Intestines

Background Escherichia coli O157:H7 strain 86–24 grown in MacConkey broth (MB) shows almost no adherence to cultured epithelial cells but adheres well in pig ligated intestines. This study investigated the mechanisms associated with the difference between in-vitro and in-vivo adherence of the MB culture.Methodology/Principal FindingsIt was found that decreased adherence in vitro by bacteria grown in MB was mainly due to lactose, possibly implicating the involvement of carbon catabolite repression (CCR). Expression of selected virulence-related genes associated with adherence and CCR was then examined by quantitative PCR. When bacteria were grown in MB and Brain Heart Infusion with NaHCO3 (BHIN) plus lactose, pH was reduced to 5.5–5.9 and there was a significant decrease in expression of the locus of enterocyte effacement (LEE) genes eae, tir, espD, grlA/R and ler, and an increase in cya (cAMP), and two negative regulators of the LEE, gadE and hfq. Putative virulence genes stcE, hlyA, ent and nleA were also decreased in vitro. Reversal of these changes was noted for bacteria recovered from the intestine, where transcripts for qseF and fis and putative virulence factors AidA15, TerC and Ent/EspL2 were significantly increased, and transcripts for AIDA48, Iha, UreC, Efa1A, Efa1B, ToxB, EhxA, StcE, NleA and NleB were expressed at high levels.Conclusions/SignificancePresence of lactose resulted in decreased expression of LEE genes and the failure of EHEC O157:H7 to adhere to epithelial cells in vitro but this repression was overcome in vivo. CCR and/or acidic pH may have played a role in repression of the LEE genes. Bacterial pathogens need to integrate their nutritional metabolism with expression of virulence genes but little is known of how this is done in E. coli O157:H7. This study indicates one aspect of the subject that should be investigated further.

Atypical Enteropathogenic Escherichia coli That Contains Functional Locus of Enterocyte Effacement Genes Can Be Attaching-and-Effacing Negative in Cultured Epithelial Cells

Enteropathogenic Escherichia coli (EPEC) induces a characteristic histopathology on enterocytes known as the attaching-and-effacing (A/E) lesion, which is triggered by proteins encoded by the locus of enterocyte effacement (LEE). EPEC is currently classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence or absence of the EPEC adherence factor plasmid, respectively. Here we analyzed the LEE regions of three aEPEC strains displaying the localized adherence-like (LAL), aggregative adherence (AA), and diffuse adherence (DA) patterns on HEp-2 cells as well as one nonadherent (NA) strain. The adherence characteristics and the ability to induce A/E lesions were investigated with HeLa, Caco-2, T84, and HT29 cells. The adherence patterns and fluorescent actin staining (FAS) assay results were reproducible with all cell lines. The LEE region was structurally intact and functional in all strains regardless of their inability to cause A/E lesions. An EspF(U)-expressing plasmid (pKC471) was introduced into all strains, demonstrating no influence of this protein on either the adherence patterns or the capacity to cause A/E of the adherent strains. However, the NA strain harboring pKC471 expressed the LAL pattern and was able to induce A/E lesions on HeLa cells. Our data indicate that FAS-negative aEPEC strains are potentially able to induce A/E in vivo, emphasizing the concern about this test for the determination of aEPEC virulence. Also, the presence of EspF(U) was sufficient to provide an adherent phenotype for a nonadherent aEPEC strain via the direct or indirect activation of the LEE4 and LEE5 operons.

The muramidase EtgA from enteropathogenic Escherichia coli is required for efficient type III secretion

Enteropathogenic Escherichia coli (EPEC) is an important cause of infectious diarrhoea. It colonizes human intestinal epithelial cells by delivering effector proteins into the host cell cytoplasm via a type III secretion system (T3SS) encoded within the chromosomal locus of enterocyte effacement (LEE). The LEE pathogenicity island also encodes a lytic transglycosylase (LT) homologue named EtgA. In the present work we investigated the significance of EtgA function in type III secretion (T3S). Purified recombinant EtgA was found to have peptidoglycan lytic activity in vitro. Consistent with this function, signal peptide processing and bacterial cell fractionation revealed that EtgA is a periplasmic protein. EtgA possesses the conserved glutamate characteristic of the LT family, and we show here that it is essential for enzymic activity. Overproduction of EtgA in EPEC inhibits bacterial growth and induces cell lysis unless the predicted catalytic glutamate is mutated. An etgA mutant is attenuated for T3S, red blood cell haemolysis and EspA filamentation. BfpH, a plasmid-encoded putative LT, was not able to functionally replace EtgA. Overall, our results indicate that the muramidase activity of EtgA is not critical but makes a significant contribution to the efficiency of the T3S process.

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

Enteropathogenic and enterohaemorrhagic Escherichia coli are related pathotypes of bacteria that cause acute watery diarrhoea and haemorrhagic colitis, respectively, and enterohaemorrhagic E. coli can lead to a serious complication known as haemolytic uraemic syndrome. In both bacteria the global regulatory protein Ler controls virulence. The ler gene is found within the locus of enterocyte effacement, or LEE, encoding a type III secretion system necessary for injecting effector proteins into intestinal epithelial cells and causing net secretory diarrhoea. The nucleoid-associated protein H-NS silences, whereas Ler serves as an anti-silencer of, multiple LEE operons. Although Ler has a higher affinity for DNA than does H-NS, the precise molecular mechanism by which Ler increases LEE transcription remains to be determined. In this report we investigate the oligomerization activity of Ler. In solution, Ler forms dimers and soluble aggregates of up to 5000 kDa molecular mass, and appears to oligomerize more readily than the related protein H-NS. An insertional mutation into the Ler linker region diminished oligomerization activity. Despite being proteins of similar mass and having homologous DNA-binding domains, Ler and H-NS complexed to DNA migrated to distinct locations, as determined by an electrophoretic mobility shift assay, implying that the related proteins form different 3D shapes in the presence of DNA. Lastly, we present electron microscopy images of toroidal Ler-DNA structures that are predicted to be involved in stimulating gene expression.

The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili ( pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.