EspP, a Type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however the molecular mechanisms that promote persistent colonization of the bovine intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes several putative virulence factors. Here, it is reported that the pO157-encoded Type V-secreted serine protease EspP influences the intestinal colonization of calves. To dissect the basis of attenuation, a bovine primary rectal epithelial cell line was developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by espP mutation but restored upon addition of highly purified exogenous EspP. Data of this study add to the growing body of evidence that cytotoxins facilitate intestinal colonization by EHEC.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7

The eae (Escherichia coli attaching and effacing) gene from enteropathogenic Escherichia coli (EPEC) was previously shown to be essential for production of the 'attaching and effacing' histopathology characteristic of EPEC infections (Jerse et al., 1990). We have now cloned the eae gene from enterohaemorrhagic E. coli (EHEC) which, in addition to producing Shiga-like cytotoxins, also produces the attaching and effacing effect. The sequence homology between the EPEC and EHEC sequences was 86% and 83% at the nucleotide and amino acid levels, respectively. The predicted amino acid sequence of the EHEC eae gene shared 31% identity and 51% similarity with invasin of Yersinia pseudotuberculosis. Alignment of the EPEC and EHEC Eae proteins and the Y. pseudotuberculosis and Y. enterocolitica invasins shows striking regions of identity with the greatest divergence at the C-terminal end, the putative receptor-binding portion of invasin.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.     

Properties of a novel periplasmic catalase-peroxidase from Escherichia coli O157:H7

A subset of catalase-peroxidases are distinguished by their periplasmic location and their expression by pathogens. Kinetic and spectral properties have not been reported for any of these enzymes. We report the cloning, expression, isolation, and characterization of KatP, a periplasmic catalase-peroxidase from Escherichia coli O157:H7. Absorption spectra indicated a mixture of heme states dominated by the pentacoordinate and hexacoordinate high-spin forms. Apparent k(cat) values for catalase (1.8x10(4) s(-1)) and peroxidase (77 s(-1)) activities were greater than those of other catalase-peroxidases. However, apparent K(M) values for H2O2 were also higher (27 mM for catalase and 3 mM for peroxidase). Ferric KatP reacted with peracetic acid to form compound I (8.8x10(3) M(-1) s(-1)) and with CN(-) to form a ferri-cyano complex (3.9x10(5) M(-1) s(-1)) consistent with other catalase-peroxidases. The isolation and characterization of KatP opens new avenues to explore mechanisms by which the periplasmic catalase-peroxidases may contribute to bacterial virulence.     

Characterization of the StcE protease activity of Escherichia coli O157:H7

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.     

Analysis of fimbrial gene clusters and their expression in enterohaemorrhagic Escherichia coli O157:H7

The sequence of two enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains reveals the possession of at least 16 fimbrial gene clusters, many of the chaperone/usher class. The first part of this study examined the distribution of these clusters in a selection of EHEC/EPEC (enteropathogenic E. coli) serotypes to determine if any were likely to be unique to E. coli O157:H7. Six of the clusters, as determined by the presence of amplified main subunit or usher gene sequences, were detected only in the E. coli O157 and O145 serotypes tested. With the exception of one serotype O103 strain that contained an lpf2 cluster, lpf sequences were only detected in E. coli O157 of the serotypes tested. Expression from each cluster was measured by the construction of chromosomally integrated lacZ promoter fusions and plasmid-based eGFP fusions in E. coli O157:H7. This analysis demonstrated that the majority (11/15) of main fimbrial subunit genes were not expressed under the majority of conditions tested in vitro. One of the clusters showing promoter activity, loc8, has a temperature expression optimum indicating a possible role outside the host. From the presence of pseudogenes in three of the clusters, the lack of FimH-like minor adhesins in the clusters and their limited expression in vitro, it would appear that E. coli O157:H7 has a limited repertoire of expressed functional fimbriae. This restricted selection of fimbriae may be important in bringing about the tropism E. coli O157:H7 demonstrates for the terminal rectum of cattle.     

Escherichia coli O157:H7 Typing Phage V7 Is a T4-Like Virus

The complete genome sequence of the Escherichia coli O157:H7 typing phage V7 was determined. Its double-stranded DNA genome is 166,452 bp long, encoding 273 proteins and including 11 tRNAs. This virus belongs to the genus T4-like viruses within the subfamily Tevenvirinae, family Myoviridae.     

Characterization of a novel two-partner secretion system in Escherichia coli O157:H7

Gram-negative bacteria contain multiple secretion pathways that facilitate the translocation of proteins across the outer membrane. The two-partner secretion (TPS) system is composed of two essential components, a secreted exoprotein and a pore-forming beta barrel protein that is thought to transport the exoprotein across the outer membrane. A putative TPS system was previously described in the annotation of the genome of Escherichia coli O157:H7 strain EDL933. We found that the two components of this system, which we designate OtpA and OtpB, are not predicted to belong to either of the two major subtypes of TPS systems (hemolysins and adhesins) based on their sequences. Nevertheless, we obtained direct evidence that OtpA and OtpB constitute a bona fide TPS system. We found that secretion of OtpA into the extracellular environment in E. coli O157:H7 requires OtpB and that when OtpA was produced in an E. coli K-12 strain, its secretion was strictly dependent on the production of OtpB. Furthermore, using OtpA/OtpB as a model system, we show that protein secretion via the TPS pathway is extremely rapid.     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Derivation of Escherichia coli O157:H7 from Its O55:H7 Precursor

There are 29 E. coli genome sequences available, mostly related to studies of species diversity or mode of pathogenicity, including two genomes of the well-known O157:H7 clone. However, there have been no genome studies of closely related clones aimed at exposing the details of evolutionary change. Here we sequenced the genome of an O55:H7 strain, closely related to the major pathogenic O157:H7 clone, with published genome sequences, and undertook comparative genomic and proteomic analysis. We were able to allocate most differences between the genomes to individual mutations, recombination events, or lateral gene transfer events, in specific lineages. Major differences include a type II secretion system present only in the O55:H7 chromosome, fewer type III secretion system effectors in O55:H7, and 19 phage genomes or phagelike elements in O55:H7 compared to 23 in O157:H7, with only three common to both. Many other changes were found in both O55:H7 and O157:H7 lineages, but in general there has been more change in the O157:H7 lineages. For example, we found 50% more synonymous mutational substitutions in O157:H7 compared to O55:H7. The two strains also diverged at the proteomic level. Mutational synonymous SNPs were used to estimate a divergence time of 400 years using a new clock rate, in contrast to 14,000 to 70,000 years using the traditional clock rates. The same approaches were applied to three closely related extraintestinal pathogenic E. coli genomes, and similar levels of mutation and recombination were found. This study revealed for the first time the full range of events involved in the evolution of the O157:H7 clone from its O55:H7 ancestor, and suggested that O157:H7 arose quite recently. Our findings also suggest that E. coli has a much lower frequency of recombination relative to mutation than was observed in a comparable study of a Vibrio cholerae lineage.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.     

Cardiovascular disease after Escherichia coli O157:H7 gastroenteritis

Background:

Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, which can be devastating in outbreak situations. We studied the risk of cardiovascular disease following such an outbreak in Walkerton, Ontario, in May 2000.

Methods:

In this community-based cohort study, we linked data from the Walkerton Health Study (2002–2008) to Ontario’s large healthcare databases. We included 4 groups of adults: 3 groups of Walkerton participants (153 with severe gastroenteritis, 414 with mild gastroenteritis, 331 with no gastroenteritis) and a group of 11 263 residents from the surrounding communities that were unaffected by the outbreak. The primary outcome was a composite of death or first major cardiovascular event (admission to hospital for acute myocardial infarction, stroke or congestive heart failure, or evidence of associated procedures). The secondary outcome was first major cardiovascular event censored for death. Adults were followed for an average of 7.4 years.

Results:

During the study period, 1174 adults (9.7%) died or experienced a major cardiovascular event. Compared with residents of the surrounding communities, the risk of death or cardiovascular event was not elevated among Walkerton participants with severe or mild gastroenteritis (hazard ratio [HR] for severe gastroenteritis 0.74, 95% confidence interval [CI] 0.38–1.43, mild gastroenteritis HR 0.64, 95% CI 0.42–0.98). Compared with Walkerton participants who had no gastroenteritis, risk of death or cardiovascular event was not elevated among participants with severe or mild gastroenteritis.

Interpretation:

There was no increase in the risk of cardiovascular disease in the decade following acute infection during a major E. coli O157:H7 outbreak.Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, causing 63 000 infections each year and 12 major outbreaks since 2006 in the United States alone.^1,2 This strain was most recently implicated in the outbreak involving beef from XL Foods (September 2012), with 17 confirmed cases across Canada.³ A similar enterohemorrhagic strain E. coli O104:H4 was responsible for an outbreak in Germany in May 2011, causing 3792 cases of gastroenteritis and 43 deaths.^4,5Most patients fully recover from acute gastroenteritis caused by E. coli. However, such an illness may predispose patients to long-term disease. Shiga toxin is produced by E. coli O157:H7; this toxin damages the microvasculature of the kidneys leading to hypertension^6–13 and directly damages the systemic vasculature.^14–16 Infected people may progress from a state of acute inflammation of the vasculature to subclinical chronic inflammation, which could promote atherosclerosis.^17–20In Walkerton, Ontario, in May 2000, heavy rains transported bovine fecal matter into the town’s well, contaminating the inadequately chlorinated municipal water supply with E. coli O157:H7.²¹ Over 2300 people developed acute gastroenteritis, and 7 people died.²² The unique circumstances of this outbreak provided a rare opportunity to study the natural history following exposure to this pathogen in a single cohort.²³ Other outbreaks have been geographically dispersed, making it difficult to track cases.^24,25In Walkerton, affected individuals were followed annually in a clinic to assess their long-term outcomes (Walkerton Health Study, 2002–2008). We previously reported that adults who experienced acute gastroenteritis during the outbreak had a higher than expected incidence of hypertension, chronic kidney disease and self-reported cardiovascular disease in follow-up.²³ However, 46% of participants were lost to follow-up by the end of the study, and there were limitations associated with the assessment of cardiovascular disease by participant recall. Thus, we conducted an expanded and extended follow-up study, linking the Walkerton study data to Ontario’s health care databases. Our objective was to more accurately determine the 10-year risk of major cardiovascular events after exposure to E. coli O157:H7.     

Inhibitory effect of creosote and its main components on production of verotoxin of enterohaemorrhagic Escherichia coli O157:H7

The inhibitory effects of creosote and its main components, alpha-methoxyphenol and o-ethylphenol, on production of verotoxin by enterohaemorrhagic Escherichia coli O157 (VTEC E. coli) were investigated. The production of verotoxin by VTEC E. coli was inhibited by the administration of 0.001-0.1% of creosote or o-ethylphenol (final concentration). On the other hand, weak inhibition of production of verotoxin was observed with 0.1% alpha-methoxyphenol administration. As the inhibitory effects were obtained below Minimal Inhibitory Concentration (MIC) values, these test compounds exerted their effects at the active site of VTEC E. coli cells prior to their production of verotoxin. These findings suggest that pre-administration of creosote and its main components might prevent human intestinal food poisoning by VTEC E. coli and contribute to maintenance of public health.     

Regulation of virulence factors of enterohemorrhagic Escherichia coli O157:H7 by self-produced extracellular factors

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes serious diarrhea and hemolytic uremic syndrome in humans. The expressions of EspD and intimin by O157:H7 have now been shown to be down-regulated by medium conditioned by O157:H7 grown at stationary phase. Preparation of conditioned medium showing the effect on the amount of EspD was not dependent on temperature or growth medium, but was dependent on growth phase. Inhibition of EspD and intimin expression was also induced by medium conditioned by E. coli K-12 strains and homoserine lactone, a signal molecule of the quorum-sensing system in gram-negative bacteria. These results suggest the possibility that the quorum-sensing system mediated by self-produced extracellular factors plays an important role in control of colonization of EHEC O157:H7.