CfaD-dependent expression of a novel extracytoplasmic protein from enterotoxigenic Escherichia coli.
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H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions -23 to -56, and the other extends from positions -73 to -103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC. (+info)
OBJECTIVE: Enterotoxigenic Escherichia coli (ETEC) is a major cause of acute diarrhoea in children in the developing world, in travellers and in the military. Safe, effective vaccines could reduce morbidity and mortality. As immunity to ETEC is strain specific, the ability to create vaccines in vitro which express multiple antigens would be desirable. It was hypothesised that three genetically attenuated ETEC strains, one with a genetic addition, would be immunogenic and safe, and they were evaluated in the first licensed UK release of genetically modified oral vaccines. METHODS: Phase 1 studies of safety and immunogenicity were carried out at a Teaching Hospital in London. Varying oral doses of any of three oral vaccines, or placebo, were administered to volunteers of both sexes (n = 98). Peripheral blood responses were measured as serum antibodies (IgG or IgA) by ELISA, antibody-secreting cell (ASC) responses by enzyme-linked immunospot (ELISPOT), and antibody in lymphocyte supernatant (ALS) by ELISA. Mucosal antibody secretion was measured by ELISA for specific IgG and IgA in whole gut lavage fluids (WGLFs). RESULTS: Significant mucosal IgA responses were obtained to colonisation factors CFA/I, CS1, CS2 and CS3, both when naturally expressed and when genetically inserted. Dose-response relationships were most clearly evident in the mucosal IgA in WGLF. Vaccines were well tolerated and did not elicit interleukin (IL) 8 or IL6 secretion in WGLF. CONCLUSIONS: Genetically modified ETEC vaccines are safe and induce significant mucosal IgA responses to important colonisation factors. Mucosal IgA responses were clearly seen in WGLF, which is useful for evaluating oral vaccines. (+info)
Part III. Analysis of data gaps pertaining to enterotoxigenic Escherichia coli infections in low and medium human development index countries, 1984-2005.
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Enterotoxigenic Escherichia coli (ETEC) is a common cause of profuse watery diarrhoea in the developing world, often leading to severe dehydration or death. We found only 15 population-based studies in low and medium human development index (HDI) countries from 1984 to 2005 that evaluate disease incidence. Reported incidence ranged from 39 to 4460 infections/1000 persons per year. The peak incidence of ETEC appeared to occur between ages 6 and 18 months. A median of 14% (range 2-36%) of diarrhoeal specimens were positive for ETEC in 19 facility- and population-based studies conducted in all age groups and 13% (range 3-39%) in 51 studies conducted in children only. Heat-labile toxin (LT)-ETEC is thought to be less likely to cause disease than heat-stable toxin (ST)-ETEC or LT/ST-ETEC. Because population-based studies involve enhanced clinical management of patients and facility-based studies include only the most severe illnesses, reliable data on complications and mortality from ETEC infections was unavailable. To reduce gaps in the current understanding of ETEC incidence, complications and mortality, large population-based studies combined with facility-based studies covering a majority of the corresponding population are needed, especially in low-HDI countries. Moreover, a standard molecular definition of ETEC infection is needed to be able to compare results across study sites. (+info)
Dietary inclusion of colicin e1 is effective in preventing postweaning diarrhea caused by F18-positive Escherichia coli in pigs.
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With worldwide concern over the use of antibiotics in animal agriculture and their contribution to the spread of antibiotic resistance, alternatives to conventional antibiotics are needed. Previous research in our laboratories has shown that colicin E1 is effective against some Escherichia coli strains responsible for postweaning diarrhea (PWD) in vitro. In this study we examined the efficacy of the dietary inclusion of colicin E1 in preventing experimentally induced PWD caused by F18-positive enterotoxigenic E. coli in young pigs. Twenty-four weaned pigs (23 days of age), identified by genotyping to be susceptible to F18-positive E. coli infections, were individually housed and fed diets containing 0, 11, or 16.5 mg colicin E1/kg diet. Two days after the start of the trial, all animals were orally inoculated with 1 x 10(9) CFU of each of two F18-positive E. coli strains isolated from pigs with PWD. The dietary inclusion of colicin E1 decreased the incidence and severity of PWD caused by F18-positive enterotoxigenic E. coli and improved the growth performance of the piglets. Additionally, the reduced incidence of PWD due to dietary colicin E1, lowered the levels of expression of the genes for interleukin 1beta and tumor necrosis factor beta in ileal tissues from these animals. The dietary inclusion of colicin E1 may be an effective alternative to conventional antibiotics in the diets of weaning pigs for the prevention of PWD caused by F18-positive enterotoxigenic E. coli. (+info)
Comparison between O serotyping method and multiplex real-time PCR to identify diarrheagenic Escherichia coli in Taiwan.
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To compare the diarrheagenic Escherichia coli (DEC) identifications obtained between traditional O serotyping and modern virulence gene detection assays, we developed a multiplex real-time PCR assay by detecting six specific virulence genes for enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). Among 261 clinical diarrheal stool samples, a total of 137 suspected DEC (sDEC) isolates were identified by the use of commercially available antisera. The most prevalent serogroups were O1 (12/137; 8.7%), O25 (9/137; 6.5%), and O44 (9/137; 6.5%). The specific virulence genes for the 137 sDEC isolates were analyzed by the multiplex real-time PCR assay. Fifteen (10.9%) of 137 isolates were confirmed to be true DEC strains, indicating that the serotypic markers did not correlate with the specific virulence genes. ETEC (66.7%) was the most prevalent, followed by EIEC (20%) and EPEC (13.3%). No EHEC strains were identified in the specimens. Four novel serotypes were found in the study: two in EPEC strains (O111:H9 and O63:H6) and two in EIEC strains (O63:H9 and O169:H9). In conclusion, the real-time PCR assay considerably reduces the high false-positive rate from the use of serotyping alone, and thus, it is suggested that serogrouping-based methods are inadequate for the identification of DEC isolates, although they are useful for the identification of a limited number of serogroups. In addition, ETEC, EPEC, and EIEC strains were present in 5.7% (15/261) of the diarrheal patients in northern Taiwan in 2006. (+info)
Deletion mutations in N-terminal alpha1 helix render heat labile enterotoxin B subunit susceptible to degradation.
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Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal alpha1 helix lies outside the core structure. However, the function of the N-terminal alpha1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the alpha1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2-7 from the N terminus, including 4 aa from the alpha1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the alpha1 helix also resulted in loss of expression. However, a single amino acid deletion outside the alpha1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcription-translation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription-translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants. (+info)
Genetic diversity of the gene cluster encoding longus, a type IV pilus of enterotoxigenic Escherichia coli.
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Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer. (+info)
Influence of geographical origin, host animal and stx gene on the virulence characteristics of Escherichia coli O26 strains.
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The influence of geographical origin, host animal and presence of the stx gene on the virulence of Escherichia coli O26 strains from ruminants was determined in this study. A clear association was found between the virulence profile and geographical origin of Shiga-toxigenic E. coli (STEC) O26 strains, with UK STEC O26 strains harbouring virtually identical profiles, whilst central European strains showed considerable heterogeneity in plasmid-encoded genes. The former group were also more likely to be non-motile and katP gene positive. Comparison of UK STEC and atypical enteropathogenic E. coli (aEPEC) O26 strains showed that the presence of the stx1 gene was positively correlated with the presence of espP and katP genes and negatively associated with the presence of the yagP-yagT region and with rhamnose fermentation. In contrast to the uniform profiles of STEC O26 strains from ruminants in the UK, aEPEC O26 strains of bovine and ovine origin showed diverse profiles both within and between groups, and could not be separated into discrete groups. These results indicate that the characteristics of UK O26 strains from ruminants are distinct from those of O26 strains from ruminants and humans in other regions in central Europe. Such differences are expected to influence the zoonotic potential of this pathogen and the subsequent incidence of O26-associated human disease. (+info)