Strain EDL933 Research Articles

The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

BackgroundEnterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).Methodology/Principal FindingsIn this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-α, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-α, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with ≥50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-α are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.Conclusions/SignificanceThe HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-α release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen.

High-throughput Sorting of an Anticalin Library via EspP-mediated Functional Display on the Escherichia coli Cell Surface

We demonstrate that small engineered single-chain binding proteins based on the lipocalin scaffold, so-called Anticalins, can be functionally displayed on the Gram-negative bacterial cell envelope. To this end, the β-domains of five different bacterial autotransporters (the IgA protease from Neisseria gonorrhoeae, the esterase EstA from Pseudomonas aeruginosa, the YpjA autotransporter from E. coli K12, the AIDA-I adhesin from enteropathogenic E. coli O127:H27 strain 2787 and the protease EspP from enterohemorrhagic E. coli O157:H7 strain EDL933) were compared with respect to display level, functional variance, and bacterial cell viability. Use of the EspP autotransporter led to a system with high genetic stability for the display of fully functional Anticalins in high density on the cell surface of E. coli as shown by quantitative flow cytofluorimetry. This system was applied to engineer an immunostimulatory Anticalin that binds and blocks the extracellular region of human CTLA-4 to achieve a slower dissociation rate. A combinatorial library of the original Anticalin was generated by error-prone PCR, subjected to E. coli cell surface display, and applied to repeated cycles of cell sorting after incubation with the fluorescently labelled target protein under competition with the unlabelled extracellular domain of CTLA-4. The resulting Anticalin variants, which were expressed and purified as soluble proteins, showed more than eightfold decelerated target dissociation, as revealed by real time surface plasmon resonance analysis. Hence, the EspP autotransporter-mediated E. coli surface display in combination with high-throughput fluorescence-activated cell sorting (FACS) provides an efficient strategy to select for Anticalins, and possibly other small protein scaffolds, with improved binding properties, which is particularly useful for in vitro affinity maturation but may also serve for the selection of novel target specificity from naive libraries.

Genome-wide transposon mutagenesis reveals a role for pO157 genes in biofilm development in Escherichia coli O157:H7 EDL933.

Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.

Identification of prophage gene z2389 in Escherichia coli EDL933 encoding a DNA cytosine methyltransferase for full protection of NotI sites

Pulsed-field gel electrophoresis (PFGE) analysis revealed that the genomes of some pathogenic Escherichia coli O157:H7 strains, including EDL933, were resistant to NotI digestion. An amino acid sequence comparison suggested that the z2389 gene carried on prophage CP-933R in strain EDL933 is likely to encode a C 5-cytosine methyltransferase. The z2389-equivalent gene was found in the NotI-resistant strains tested, but it was not detected in the NotI-susceptible strains. PFGE analysis of the wild-type EDL933 strain and of a z2389 null mutant revealed that z2389 was associated with full genome protection against NotI digestion and partial protection against EagI digestion. In vitro methylation experiments with purified recombinant protein demonstrated that Z2389 is capable of methylating NotI and EagI sites. Sequencing of bisulfite-treated DNA indicated that the methylation occurred at the first cytosine residue of the NotI recognition sequence, whereas EagI sites remained unmethylated or were methylated at the first cytosine residue. Thus, z2389 encodes a DNA cytosine methyltransferase that confers full protection to NotI sites.

Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains

Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx(2) flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx(2)) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx(2), whereas all LII strains carried variant stx(2c) and 4 of 14 LI/II strains had copies of both stx(2) and variant stx(2c). Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx(2) mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

Differential effects of short-chain fatty acids and iron on expression of iha in Shiga-toxigenic Escherichia coli

Shiga-toxigenic Escherichia coli (STEC) colonizing the bowel are exposed to a variety of short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, produced by gut microflora. However, the total concentrations and relative amounts of SCFAs in the lumen vary with intestinal niche. Here we report that conditions simulating SCFA concentrations present in the human gut trigger expression of the iha gene, which encodes an adherence-conferring outer-membrane protein of pathogenic E. coli. We show that growth under conditions simulating colonic, but not ileal, SCFA concentrations increases iha expression in three tested STEC strains, with the strongest expression detected in LEE-negative STEC O113:H21 strain 98NK2. Expression of iha is known to be subject to Fur-mediated iron repression in O157:H7 STEC, and the same occurs in 98NK2. However, exogenous iron did not repress iha expression in the presence of colonic SCFAs in either 98NK2 or the O157:H7 strain EDL933. Moreover, exposure to the iron chelator 2,2'-dipyridyl caused no further enhancement of iha expression over that induced by colonic SCFAs. These findings indicate that SCFAs regulate iha expression in STEC independently of iron. Increased expression of iha under colonic but not ileal SCFA conditions possibly may contribute to preferential colonization of the human colon by STEC.

KatP contributes to OxyR-regulated hydrogen peroxide resistance in Escherichia coli serotype O157 : H7

Escherichia coli K-12 defends itself against peroxide-mediated oxidative damage using two catalases, KatG and KatE, and the peroxiredoxin, alkyl hydroperoxide reductase, encoded by ahpC. In E. coli O157 : H7 strain ATCC 43895 (EDL933), plasmid pO157 carries an additional catalase-peroxidase gene, katP. KatP has been shown to be a functional catalase-peroxidase. However, deletion of pO157 does not alter the peroxide resistance of strain EDL933, leaving the physiological role of katP unclear. To examine the individual roles of peroxide-resistance genes in E. coli O157 : H7, mutant strains of ATCC 43895 were constructed bearing individual deletions of katG, katE, katP and ahpC, as well as double, triple and quadruple deletions encompassing all possible gene combinations thereof. The wild-type and all 15 mutant strains were compared for differences in aerobic growth, ability to scavenge exogenous H(2)O(2) and resistance to exogenous peroxides. Although KatG scavenged the most exogenous H(2)O(2), KatP scavenged statistically greater amounts than either KatE or AhpC during exponential growth. However, katG and ahpC together were sufficient for full peroxide resistance in disc diffusion assays. Strains with only katG or ahpC were the only triple deletion strains with significantly shorter generation times than the quadruple deletion strain. ahpC was the only gene that could allow rapid transition from lag phase to exponential phase in a triple deletion strain. Gene expression studies revealed that katP is an OxyR-regulated gene, but its expression is suppressed in stationary phase by RpoS. These studies indicate that pO157-borne katP contributes to the complex gene network protecting strain 43895 from peroxide-mediated oxidative damage in an OxyR-dependent manner.

Towards an attenuated enterohemorrhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/ stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.

The Escherichia coli ycbQRST operon encodes fimbriae with laminin-binding and epithelial cell adherence properties in Shiga-toxigenic E. coli O157:H7.

The human pathogen Shiga-toxigenic Escherichia coli (STEC) O157:H7 contains a ycbQRST fimbrial-like operon, which shares significant homology to the family of F17 fimbrial biogenesis genes f17ADCG found in enterotoxigenic E. coli. We report that growth of STEC O157:H7 strain EDL933 in minimal Minca medium at 37°C and during adherence to epithelial cells led to the production of fine peritrichous fimbriae, which were found to be composed of a major subunit of 18 kDa whose N-terminal amino acid sequence matched the predicted protein product of the ycbQ gene; and showed significant homology to the F17a-A fimbrin. Similar to the F17 fimbriae, the purified STEC fimbriae and the recombinant YcbQ protein fused to a His peptide tag bound laminin, but not fibronectin or collagen. Thus, we propose the name E. coli YcbQ laminin-binding fimbriae (ELF) to designate the fimbriae encoded by the ycbQRST operon. The role of ELF as an adherence factor of STEC to cultured epithelial cells was investigated. We provide compelling evidence demonstrating that ELF contributes to adherence of STEC to human intestinal epithelial cells and to cow and pig gut tissue in vitro. Deletion in the fimbrin subunit gene elfA (or ycbQ) in STEC strain EDL933 led to an isogenic strain, which showed significant reduction (60%) in adherence to HEp-2 cells in comparison with the parental strain. In addition, antibodies against the purified ELF also partially blocked adherence of two STEC O157:H7 strains. These observations suggest that ELF functions as an accessory adherence factor that, along with other known redundant adhesins, contributes to the overall adhesive properties of STEC O157:H7 providing these organisms with ecological advantages to survive in different hosts and in the environment.

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E. coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E. coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E. coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E. coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.

Transcriptome analysis ofEscherichia coli O157:H7 EDL933 during heat shock

The response to elevated temperature [heat shock (HS)] is highly conserved. The transcriptome of Escherichia coli K-12 has been studied under a variety of conditions while such studies involving E. coli O157:H7 are only now being conducted. To better understand the impact of HS on E. coli O157:H7, global transcript levels of strain EDL933 cells shifted from 37 to 50 degrees C for 15 min were compared with cells held at 37 degrees C by microarray. Using a mixed model analysis, 193 genes were found to be differentially transcribed at P<0.0042 with a q value <0.1. The 111 downregulated genes include the curli pili-associated genes csgABCDEFG, maltose transport-associated proteins malEFK, and NADH dehydrogenase subunit encoding nuoCEHIJN. The 82 genes upregulated include the HS-induced genes rpoH, dnaK, dnaJ, groEL, groES, and grpE along with two LEE-encoded genes: hypothetical gene Z5121 and sepZ. Twenty-three additional genes located in O-islands were found to be differentially expressed. Quantitative real-time PCR (qRT-PCR) was performed to validate the microarray results. Also, samples subjected to a 30-42 degrees C shift were examined by qRT-PCR to confirm differential transcription of selected genes. These results indicate that this pathogen may regulate its virulence factors in response to temperature changes.

Characterization of the α-haemolysin determinant from the human enteropathogenicEscherichia coliO26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding alpha-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire alpha-hlyCABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited alpha-hly determinants. The alpha-hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli alpha-hly plasmid pHly152, in particular, the structural alpha-hlyCABD genes (99.2% identity) and the regulatory hlyR regions (98.8% identity). pEO5 and alpha-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural alpha-hlyCABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS2 element upstream of the hlyC gene in pHly152. The presence of transposon-like structures at both ends of the alpha-hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.

Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933

BackgroundRpoS is a conserved stress regulator that plays a critical role in survival under stress conditions in Escherichia coli and other γ-proteobacteria. RpoS is also involved in virulence of many pathogens including Salmonella and Vibrio species. Though well characterized in non-pathogenic E. coli K12 strains, the effect of RpoS on transcriptome expression has not been examined in pathogenic isolates. E. coli O157:H7 is a serious human enteropathogen, possessing a genome 20% larger than that of E. coli K12, and many of the additional genes are required for virulence. The genomic difference may result in substantial changes in RpoS-regulated gene expression. To test this, we compared the transcriptional profile of wild type and rpoS mutants of the E. coli O157:H7 EDL933 type strain.ResultsThe rpoS mutation had a pronounced effect on gene expression in stationary phase, and more than 1,000 genes were differentially expressed (twofold, P < 0.05). By contrast, we found 11 genes expressed differently in exponential phase. Western blot analysis revealed that, as expected, RpoS level was low in exponential phase and substantially increased in stationary phase. The defect in rpoS resulted in impaired expression of genes responsible for stress response (e.g., gadA, katE and osmY), arginine degradation (astCADBE), putrescine degradation (puuABCD), fatty acid oxidation (fadBA and fadE), and virulence (ler, espI and cesF). For EDL933-specific genes on O-islands, we found 50 genes expressed higher in wild type EDL933 and 49 genes expressed higher in the rpoS mutants. The protein levels of Tir and EspA, two LEE-encoded virulence factors, were elevated in the rpoS mutants under LEE induction conditions.ConclusionOur results show that RpoS has a profound effect on global gene expression in the pathogenic strain O157:H7 EDL933, and the identified RpoS regulon, including many EDL933-specific genes, differs substantially from that of laboratory K12 strains.

Subtractive hybridization and identification of putative adhesins in a Shiga toxin-producing eae-negative Escherichia coli

Adherence to epithelial cells by specific adhesins is a characteristic of Shiga toxin-producing Escherichia coli (STEC) strains. The eae-encoded protein intimin is the main adhesin implicated in intestinal colonization in vivo. We recently showed that STEC strains isolated in Chile displayed a wide variety of adhesins; here we demonstrate that some of these STEC strains are eae-negative and still adhere to epithelial cells at a level 100-fold higher than enterohaemorrhagic E. coli (EHEC) O157 : H7 prototype strain EDL933. This phenotype is associated with the presence of adherence factors different from the intimin protein. Subtractive hybridization between EHEC EDL933 and STEC eae-negative strain 472-1 was used to identify regions implicated in adhesion. In addition to the saa gene, we identified 18 specific genes in STEC 472-1, 16 of which had nucleotide identity to Salmonella ST46 phage genes; the two remaining ones shared identity to a gene encoding a hypothetical protein of uropathogenic E. coli. The DNA sequence of the STEC 472-1 psu-int region identified five open reading frames with homology to phage genes. We constructed mutant strains in the saa gene and the psu-int region to study the participation of these genes in the adherence to epithelial cells and our results demonstrated that STECDeltasaa and STECDeltapsu-int mutants displayed a 10-fold decrease in adherence as compared to the STEC 472-1 wild-type strain. Overall, our results suggest that STEC strain 472-1 adheres to epithelial cells in an eae-independent matter and that saa and psu-int participate in this adhesion process.

Escherichia coliO157:H7 Survives within Human Macrophages: Global Gene Expression Profile and Involvement of the Shiga Toxins

Escherichia coli O157:H7 is an important food-borne pathogen that specifically binds to the follicle-associated epithelium in the intestine, which rapidly brings this bacterial pathogen in contact with underlying human macrophages. Very little information is available about the interaction between E. coli O157:H7 and human macrophages. We evaluated the uptake and survival of strain EDL933 during infection of human macrophages. Surprisingly, EDL933 survived and multiplied in human macrophages at 24 h postinfection. The global gene expression profile of this pathogen during macrophage infection was determined. Inside human macrophages, upregulation of E. coli O157:H7 genes carried on O islands (such as pagC, the genes for both of the Shiga toxins, and the two iron transport system operons fit and chu) was observed. Genes involved in acid resistance and in the SOS response were upregulated. However, genes of the locus of enterocyte effacement or genes involved in peroxide resistance were not differentially expressed. Many genes with putative or unknown functions were upregulated inside human macrophages and may be newly discovered virulence factors. As the Shiga toxin genes were upregulated in macrophages, survival and cytotoxicity assays were performed with isogenic Shiga toxin mutants. The initial uptake of Shiga toxins mutants was higher than that of the wild type; however, the survival rates were significantly lower at 24 h postinfection. Thus, Shiga toxins are implicated in the interaction between E. coli O157:H7 and human macrophages. Understanding the molecular mechanisms used by E. coli to survive within macrophages may help in the identification of targets for new therapeutic agents.

RpoS, H-NS, and DsrA influence EHEC hemolysin operon (ehxCABD) transcription inEscherichia coliO157:H7 strain EDL933

Escherichia coli hemolysin, also termed alpha-hemolysin, is a virulence factor for extraintestinal E. coli. Hemolysin operon (hlyCABD) transcription is inhibited by the nucleoid-associated protein, H-NS. This inhibition is stronger at lower growth temperature. This study investigated transcription of the homologous EHEC hemolysin (enterohemolysin) operon (ehxCABD) in EHEC O157:H7 strain EDL933. We examined the influence of H-NS, the sigma factor RpoS, and the small RNA DsrA, which is known to inhibit H-NS function and to stimulate RpoS synthesis. During growth at 30 degrees C, DsrA overexpression increased ehxA transcription in the wild type but not in an hns deletion mutant. During growth at 37 degrees C, DsrA overexpression increased ehxA transcription independent of hns genotype. This indicates that DsrA influences ehxCABD operon transcription by two different routes, one (at lower temperature) at least partially dependent on H-NS, and one (at higher temperature) independent of H-NS. An rpoS deletion mutant expressed nondetectable levels of ehxA mRNA regardless of growth temperature or DsrA overexpression, indicating that the RpoS sigma factor is essential for ehxCABD operon expression.

A homolog of the O157 urease-encoding O island 48 is present in porcine O149:H10 enterotoxigenicEscherichia coli

The relationship of the urease operon in the highly virulent O149 porcine enterotoxigenic Escherichia coli (ETEC) strain Ro8 to a genomic island (GI) homologous to O island (OI) 48 of O157 enterohemorrhagic E. coli (EHEC) strain EDL933 was investigated. Eighty-four of 84 O149:H10 strains were urease positive whereas 44 of 44 O149:H43 porcine ETEC strains were urease-negative. Seventeen of 17 O149:H10 strains that were tested possessed the OI-48 homolog whereas 24 of 24 O149:H43 strains lacked this OI. Transposon insertions in lipB or guaA genes in strain Ro8 eliminated urease activity while insertions in the caiF gene increased urease activity. When the O149 ure operon was cloned on a high copy number plasmid, urease expression was increased approximately 11-fold in Ro8 and 83-fold in O157 strain EDL933 compared with that in the wild type Ro8. The O149 urease activity was expressed despite the presence of the same premature stop codon in ureD that is present in ure+ O157:H7 strains that are urease-negative. The ure operon in Ro8 consists of 4 893 nucleotides with 99% identity with the ure operons in EHEC O157:H7 strains EDL933 and Sakai, and is part of a GI similar to GI-48 of strain EDL933. This OI, designated OI-48149 , is inserted in the serX tRNA gene in strain Ro8 and contains genes for urease, tellurite resistance, iha and an AIDA-I-like adhesin. The presence of a homolog of the O157:H7 OI-48 in highly virulent O149 porcine ETEC suggests that this OI may contribute to establishment of the bacteria in the intestine.

EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation

Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ehaA, z0469/ehaB, z3487/ehaC and z3948/ehaD) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655flu background. Preliminary characterization revealed that ehaA, ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes (ehaA) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H(-) did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.

Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli : Distribution, Secretion, and Proteolytic Activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPalpha, espPbeta, espPgamma, and espPdelta), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPbeta, espPgamma, and espPdelta genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPalpha, espPbeta, espPgamma, and espPdelta in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPalpha (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPgamma cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPbeta and EspPdelta either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.

Optical maps distinguish individual strains of Escherichia coli O157 : H7

Optical maps of 11 Escherichia coli O157 : H7 strains have been generated by the assembly of contiguous sets of restriction fragments across their entire 5.3 to 5.6 Mbp chromosomes. Each strain showed a distinct, highly individual configuration of 500-700 BamHI fragments, yielding a map resembling a DNA 'bar code'. The accuracy of optical mapping was assessed by comparing directly the in silico restriction maps of two wholly sequenced reference genomes of E. coli O157 : H7, i.e. EDL933 and the Sakai isolate (RIMD 0509952), with the optical maps of the same strains. The optical maps of nine other E. coli O157 : H7 strains were compared similarly, using the sequence-based maps of the Sakai and EDL933 strains as references. A total of 91 changes at 28 loci were positioned and sized; these included complex chromosomal inversions, insertions, deletions, substitutions, as well as a number of simple RFLPs. The optical maps defined unique genome landmarks in each of the strains and demonstrated the ability of optical mapping to distinguish and differentiate, at the individual level, strains of this important pathogen.