CsrA and TnaB coregulate tryptophanase activity to promote exotoxin-induced killing of Caenorhabditis elegans by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli(EPEC) requires the tnaA-encoded enzyme tryptophanase and its substrate tryptophan to synthesize diffusible exotoxins that kill the nematode Caenorhabditis elegans. Here, we demonstrate that the RNA-binding protein CsrA and the tryptophan permease TnaB coregulate tryptophanase activity, through mutually exclusive pathways, to stimulate toxin-mediated paralysis and killing of C. elegans.     

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.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

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.     

Molecular evolution of typical enteropathogenic Escherichia coli: clonal analysis by multilocus sequence typing and virulence gene allelic profiling

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Typical EPEC isolates are differentiated from other types of pathogenic E. coli by two distinctive phenotypes, attaching effacement and localized adherence. The genes specifying these phenotypes are found on the locus of enterocyte effacement (LEE) and the EPEC adherence factor (EAF) plasmid. To describe how typical EPEC has evolved, we characterized a diverse collection of strains by multilocus sequence typing (MLST) and performed restriction fragment length polymorphism (RFLP) analysis of three virulence genes (eae, bfpA, and perA) to assess allelic variation. Among 129 strains representing 20 O-serogroups, 21 clonal genotypes were identified using MLST. RFLP analysis resolved nine eae, nine bfpA, and four perA alleles. Each bfpA allele was associated with only one perA allele class, suggesting that recombination has not played a large role in shuffling the bfpA and perA loci between separate EAF plasmids. The distribution of eae alleles among typical EPEC strains is more concordant with the clonal relationships than the distribution of the EAF plasmid types. These results provide further support for the hypothesis that the EPEC pathotype has evolved multiple times within E. coli through separate acquisitions of the LEE island and EAF plasmid.     

Role of EspF in host cell death induced by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries. Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island. One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function. EspF, like the other Esps, is a substrate for secretion by the type III secretory system. Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation. As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability. The espF mutant was deficient in host cell killing despite having normal adherence. The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death. The mode of cell death in cells transfected with espF appeared to be pure apoptosis. EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins.     

Systematic identification and sequence analysis of the genomic islands of the enteropathogenic Escherichia coli strain B171-8 by the combined use of whole-genome PCR scanning and fosmid mapping

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are diarrheagenic pathogens that colonize the intestinal tract through the formation of attaching and effacing lesions, induced by effectors translocated via a type III secretion system (T3SS) encoded on the locus of enterocyte effacement (LEE). In EHEC O157, numerous virulence factors, including around 40 T3SS effectors, have been identified. Most of them are encoded on genomic islands (GEIs) such as prophages and integrative elements. For EPEC, however, no systematic search of GEIs and virulence-related genes carried therein has been done, and only a limited number of virulence factors have been identified so far. In this study, we performed a systemic and genome-wide survey of the GEIs in strain B171-8, one of the prototype strains of EPEC, by the combined use of whole-genome PCR scanning and fosmid mapping and identified 22 large GEIs, including nine lambda-like prophages, three P2-like prophages, the LEE, and three additional integrative elements. On these prophages and integrative elements, we found genes for a set of T3SS proteins, a total of 33 T3SS effectors or effector homologues, and 12 other virulence factors which include five nonfimbrial adhesins. Most of the T3SS effector families identified are also present in EHEC O157, but B171-8 possesses a significantly smaller number of effectors. Not only the presence or absence of Shiga toxin genes but also the difference in the T3SS effector repertoire should be considered in analyzing the pathogenicity of EPEC and EHEC strains.     

Characterization and evidence of mobilization of the LEE pathogenicity island of rabbit-specific strains of enteropathogenic Escherichia coli

We have characterized the LEE pathogenicity islands (PAIs) of two rabbit-specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H-) and 84/110-1 (O103:H2), and have compared them to homologous loci from the human enteropathogenic and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC strain, RDEC-1. All five PAIs contain a 34 kb core region that is highly conserved in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly larger (59 540 basepairs) than those of the human strains, which are less than 44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA and senA. The LEE of 84/110-1 is approximately 85 kb and is located at pheV tRNA. Its core is almost identical to those of 83/39 and RDEC-1, apart from a larger espF gene, but its flanking regions contain trcA, a putative virulence determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int-phe. The LEE PAI of 84/110-1 is also flanked by short direct repeats (representing the 3'-end of pheV tRNA), suggesting that it may be unstable. To investigate this possibility, we constructed a LEE::sacB derivative of 84/110-1 and showed that the PAI was capable of spontaneous deletion. We also showed that Int-phe can mediate site-specific integration of foreign DNA at the pheU tRNA locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.     

A Family of Indoles Regulate Virulence and Shiga Toxin Production in Pathogenic E. coli

Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) are intestinal pathogens that cause food and water-borne disease in humans. Using biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA), and indole-3-acetic acid (IAA), as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE), which encodes virulence factors that cause “attaching and effacing” (A/E) lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling.     

Characterisation of atypical enteropathogenic E. coli strains of clinical origin

Background  

Enteropathogenic E. coli (EPEC) is a prominent cause of diarrhoea, and is characterised in part by its carriage of a pathogeniCity island: the locus for enterocyte effacement (LEE). EPEC is divided into two subtypes according to the presence of bundle-forming pili (BFP), a fimbrial adhesin that is a virulence determinant of typical EPEC (tEPEC), but is absent from atypical EPEC (aEPEC). Because aEPEC lack BFP, their virulence has been questioned, as they may represent LEE-positive Shiga toxin-producing E. coli (STEC) that have lost the toxin-encoding prophage, or tEPEC that have lost the genes for BFP. To determine if aEPEC isolated from humans in Australia or New Zealand fall into either of these categories, we undertook phylogenetic analysis of 75 aEPEC strains, and compared them with reference strains of EPEC and STEC. We also used PCR and DNA hybridisation to determine if aEPEC carry virulence determinants that could compensate for their lack of BFP.     

Relationship between phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains from diverse human and animal sources

Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx(1c) and/or stx(2d), ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.     

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions

Abstract A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli , Hafnia alvei , a strain of Citrobacter freundii , and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:H6, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii , EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii , EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eaeA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.     

Molecular Evolution of the Intimin Gene in O111 Clones of Pathogenic Escherichia coli

Intimin is an important virulence factor in two groups of enteric pathogens: enteropathogenic Escherichia coli (EPEC), which is a major cause of infant diarrhea in the developing world, and enterohemorrhagic E. coli (EHEC), which has caused large food-borne outbreaks of hemorrhagic colitis in the United States and other developed countries. Intimin is encoded on a 35-kb pathogenicity island called the locus of enterocyte effacement (LEE). At least five antigenic types have been described for the highly variable gene, and each type is generally characteristic of particular evolutionary lineages. We determined the nucleotide sequences of intimin and other LEE genes in two O111 clones that have not been amenable to typing. The sequences from both O111:H8 and O111:H9 differed from the Int-beta that is typical of other clones in the same evolutionary lineage. The sequence from the O111:H8 strains was a mosaic of divergent segments that alternately clustered with Int-alpha, Int-beta, or Int-gamma. The sequence from the O111:H9 clone consistently showed a close relationship with that from E2348/69, a distantly related strain that expresses Int-alpha. The results suggest that there have been multiple acquisitions of the LEE in the EHEC 2/EPEC 2 clonal lineage, with a recent turnover in either O111:H8 or its close relatives. Amino acid substitutions that alter residue charge occurred more frequently than would be expected under random substitution in the extracellular domains of intimin, suggesting that diversifying selection has promoted divergence in this region of the protein. An N-terminal domain that presumably functions in the periplasm may also be under positive selection.     

Identification of genetic markers for differentiation of Shiga toxin-producing, enteropathogenic, and avirulent strains of Escherichia coli O26

Shiga toxin-producing Escherichia coli (STEC) O26 is one of the top five enterohemorrhagic E. coli (EHEC) O groups most often associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. STEC O26 is considered to have evolved from enteropathogenic (EPEC) O26 strains through the acquisition of Shiga toxin (Stx)-encoding genes. Our PCR data identified several STEC-like strains expressing all features of STEC except Stx production and carrying remnants of Stx phages that were probably derivatives of EHEC O26. EHEC and EPEC O26 strains phenotypically resemble O26 EHEC-like and apathogenic E. coli O26 strains and are therefore undistinguishable by cultural methods. A clear discrimination between the different O26 groups is required for diagnostics in patients and for control of food safety. To develop an assay for specific detection of EHEC and EHEC-like O26 strains, we used a high-throughput PCR approach for selection of discriminative genetic markers among 33 tested genes mostly encoding type III secretion system effector proteins. The genes ECs1822, nleH1-2, nleA, nleC, nleH1-1, nleG, nleG2, nleG6-1, nleG6-2, espJ, espM2, nleG8-2, espG, ent (or espL2), nleB, nleE, efa1, and espB were detected at different frequencies in O26 EHEC, EHEC-like, and EPEC strains, indicating the possible role of these genes in virulence of human pathogenic O26 strains. The espK and espN genes were detected only in EHEC and EHEC-like O26 strains. espK was present in 99.14% of EHEC and 91.14% of EHEC-like O26 strains and was hence the best candidate as a genetic marker for characterizing these pathogroups. These data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. The results indicate that a combination of molecular detection tools for O26 wzx (wzx(O26)), eae-beta, stx, espK, and arcA genotyping is highly discriminative for clear identification of EHEC and EHEC-like E. coli O26 strains. This simple diagnostic test might be applicable in hospital service laboratories or public health laboratories to test strains isolated from stools of patients suffering from diarrhea.