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S Knutton

Publications and source records attributed to S Knutton.

At least 19 recordsLinked to original sources
PubMed1995

Carbon dioxide regulated secretion of the EaeB protein of enteropathogenic Escherichia coli.

An eaeA mutant (intimin deficient) of enteropathogenic Escherichia coli stimulated phosphorylation of several host cell proteins, showing that intimate adherence is not required to activate signal transduction pathways in enteropathogenic E. coli-infected cells. Growth of enteropathogenic E. coli in tissue culture medium in 5% CO2, in the presence or absence of cultured cells, resulted in the secretion of several bacterial proteins. Two of these, 36 kDa and 20 kDa in size, were expressed at significantly lower levels in air. N-terminal sequencing and analysis of secreted proteins of an eaeB mutant indicated that the 36 kDa secreted protein was EaeB, previously implicated in the stimulation of signalling pathways in enteropathogenic E. coli-infected cells.

Amino Acid Sequence↗
PubMed1994

Enteroaggregative Escherichia coli: another cause of acute and chronic diarrhoea in England?

Enteroaggregative Escherichia coli (EAggEC) has been found to be associated with acute and persistent diarrhoea in children in developing countries. Its clinical significance in developed countries has not been examined in much detail. In a survey of faecal samples from children with diarrhoea presenting to a children's hospital in East London between August and December 1988, EAggEC strains were isolated in 8 of 297 (2.7%) consecutive stool samples collected from 289 children and in 5 of 34 typed E. coli isolates during 1988. Of the 13 children found to be excreting EAggEC, 8 had acute diarrhoea and 5 had chronic diarrhoea of more than 14 days' duration; 5 children had mixed infections. Compared to other organisms found during the same period, EAggEC were isolated as frequently as many other better-known pathogens such as enteropathogenic E. coli, Campylobacter, Salmonella, Shigella, Giardia lamblia, and Cryptosporidium. We conclude that EAggEC may be an important pathogen in developed countries.

Acute Disease↗
PubMed1993

The attaching and effacing virulence property of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) remain an important cause of infant diarrhoea in many parts of the developing world. Essential for virulence is their ability to adhere to the small intestinal mucosa and produce a striking 'attaching and effacing' (AE) lesion characterised by localised destruction of brush border microvilli, intimate attachment of bacteria to the residual apical enterocyte membrane, often in a cuplike pedestal structure, and formation of a dense plaque of actin (and other) cytoskeletal filaments beneath adherent bacteria. Fluorescence actin staining (FAS test) has turned out to be a useful diagnostic test for the AE lesion and also led to the identification of a chromosomal gene, eae, which is necessary but, by itself, not sufficient to produce the AE lesion. The 94 kDa outer membrane protein encoded by eae may be the adhesin which promotes intimate bacterial attachment. The signal transduction pathway which leads to AE lesion formation has yet to be defined although EPEC induced increased levels in intracellular calcium and phosphorylation of specific cell proteins including myosin light chain suggest that EPEC, by binding to a specific host cell receptor, may be promoting a calcium second message which would a) activate the brush border protein villin to cause microvillar breakdown and b) stimulate protein kinase activity to cause the other cytoskeletal rearrangements.

Actins↗
PubMed1993

Pathological changes in the rabbit ileal loop model caused by Campylobacter jejuni from human colitis.

Four strains of Campylobacter jejuni isolated from children with inflammatory diarrhoea were assayed in the rabbit ileal loop model of infectious diarrhoea. All caused inflammatory reactions with severe macroscopic and microscopic damage in infected rabbit ileal tissue similar to that observed in the patients by endoscopy and histological analysis of colonic biopsies. Haemoglobin and other proteins were observed in loop fluids, consistent with leakage of serum from damaged mucosa. Loop fluids also contained significant bicarbonate concentrations, indicative of an active secretory component similar to that in control loops inoculated with cholera toxin. However, although three of the four clinical strains produced small amounts of a protein immunologically related to cholera toxin in vitro, none such was detected in either tissues or fluids of infected ileal loops. We propose instead that host-derived mediators of secretion may be important in pathogenesis. A mutant strain of C. jejuni with impaired motility, obtained from the National Collection of Type Cultures, did not induce tissue damage or fluid secretion in rabbit ileal loops.

Animals↗
PubMed1993

The Myf fibrillae of Yersinia enterocolitica.

The Myf antigen produced by Yersinia enterocolitica appeared as a proteic polymer composed of 21 kDa subunits. By transposon mutagenesis we isolated Myf-defective mutants. Those allowed us to clone and sequence a 4.4 kb chromosomal locus involved in Myf production. This region was found to contain three genes that we called myfA, myfB and myfC. Genes myfB and myfC encode an assembly machine related to those involved in the synthesis of many fimbriae: MyfB, the putative chaperone, possesses the consensus residues of the PapD family and myfC encodes a putative outer-membrane protein. MyfA, the major subunit, was found to be 44% identical to the pH 6 antigen of Y. pestis. Myf is thus the Y. enterocolitica counterpart of this antigen, but it is by far not so well conserved as the other virulence determinants such as the Yops, suggesting that Myf and pH 6 antigen do not necessarily play the same role in Y. enterocolitica and Y. pestis. The study of the prevalence of myfA in various species of Yersinia revealed that, like the yst enterotoxin gene, its presence is restricted to the pathogenic serotypes of Y. enterocolitica. By immunogold labelling, Myf appeared as a layer of extracellular material extending locally 2 microns from the bacterial surface, indicative of a fibrillar structure.

Amino Acid Sequence↗
PubMed1993

Calcium-calmodulin dependence of actin accretion and lethality in cultured HEp-2 cells infected with enteropathogenic Escherichia coli.

Infection of cultured HEp-2 cells with enteropathogenic Escherichia coli causes substantial actin accretion at points of bacterial contact and cell death. Loss of viability was delayed by chelating intracellular free calcium. Actin accretion was partially inhibited by preventing elevation of free cytosolic calcium and prevented by treatment with a calmodulin inhibitor.

Actins↗
PubMed1993

Evaluation of the fluorescence actin staining test for detection of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) strains designated on the basis of their serotypes are epidemiologically associated with diarrhea. They adhere to the intestinal mucosa, producing the characteristic attaching and effacing (AE) lesion in an in vitro organ culture system. EPEC manifest localized adherence (LA) in the HEp-2 cell assay, and this is commonly used for clinical diagnosis. Recently, the fluorescence actin staining (FAS) test was proposed for the identification of E. coli causing the AE lesion. We therefore compared the FAS test with the HEp-2 cell assay and the EPEC adherence factor (EAF) probe assay for the detection of EPEC strains. Among 240 stool samples from children with diarrhea examined, 176 yielded E. coli and 14 of these strains showed the LA pattern in the HEp-2 cell assay; 11 of these were positive by both the EAF and the FAS tests. By using the HEp-2 cell assay as the "gold standard," the FAS test gave a sensitivity of 78.5% and a specificity of 100%. The three localized adherent FAS-negative strains tested subsequently were positive by the enteroaggregative E. coli DNA Probe and failed to produce the AE lesions characteristic of EPEC. When these strains were not considered, the sensitivity of the FAS test for detecting isolates that manifest LA was 100%. Against the EAF probe, the sensitivity and specificity of the FAS test were 91.6 and 100%, respectively. The FAS test avoids infrequent but nevertheless important phenotypic misclassifications in the HEp-2 cell assay, and it may therefore serve as a confirmatory test for EPEC.

Actins↗
PubMed1992

Intestinal epithelial cell protein phosphorylation in enteropathogenic Escherichia coli diarrhoea.

The ability of enteropathogenic Escherichia coli (EPEC) to cause diarrhoea in man is associated with the formation of characteristic histopathological lesions in small-intestine enterocytes, with gross cytoskeletal damage and loss of brush-border microvilli. Investigation of enterocyte protein phosphorylation in response to EPEC infection showed that the major phosphorylated protein, identified by immunoprecipitation, is myosin light-chain--an important cytoskeletal protein known to affect actin organisation in non-muscle cells. High enterocyte concentrations of actin and myosin were observed at sites of bacterial infection. Our findings indicate that enterocyte cytoskeletal changes in response to EPEC may be directly triggered by bacterial adherence through signal transduction pathways that stimulate protein kinase activity.

Actins↗
PubMed1992

Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.

A collection of 44 Campylobacter isolates (37 C. jejuni and seven C. coli) from children with colitis (21 strains) or watery diarrhoea (23 strains) was analysed for toxin production, association with HeLa cells, and invasion of differentiated Caco-2 cell cultures. There was no obvious association of clinical symptoms with species, biotype or enterotoxin production. All colitis strains and most of the isolates from watery diarrhoea were cytotoxic for Chinese hamster ovary cells. Measurements of bacterial association indices with HeLa cells varied with time, and were considered to be unreliable for discriminating between isolates from the two diagnostic groups. Statistically significant differences were observed between the two groups (all colitis strains and 65% of strains from non-inflammatory diarrhoea) with respect to invasion of both HeLa and Caco-2 cell monolayers. However, among the strains from non-inflammatory diarrhoea that did invade, numbers of internalised bacteria were similar to the range observed for colitis strains. Of the colitis strains, 86% were able to transcytose through polarised Caco-2 monolayers grown on filters, compared with 48% of isolates from non-inflammatory disease. We propose the use of Caco-2 cells as a model for studying invasion of intestinal epithelia by C. jejuni and C. coli.

Animals↗
PubMed1992

Ability of enteroaggregative Escherichia coli strains to adhere in vitro to human intestinal mucosa.

A collection of 44 enteroaggregative Escherichia coli (EAggEC) strains isolated from infants with diarrhea in India and the United Kingdom were examined for their ability to adhere in vitro to human intestinal mucosa and by electron microscopy for production of putative adherence factors. None of the strains adhered to human duodenal mucosa, and six strains tested did not adhere to ileal mucosa; all 44 strains, however, adhered to human colonic mucosa in localized aggregates. Electron microscopy of infected colonic mucosa indicated fimbrially mediated adhesion of the EAggEC strains. Four morphologically distinct kinds of fimbriae, including a new morphological type of E. coli fimbriae consisting of bundles of fine filaments, were identified among the EAggEC strains; this new type of fimbria was observed in 43 of the 44 EAggEC strains. Forty-three of the 44 EAggEC strains were positive with a DNA probe developed to identify EAggEC, and most of the strains belonged to serotypes unrelated to the other major classes of diarrheic E. coli. These results suggest that EAggEC may be a large-bowel pathogen and colonize the colon by a fimbrially mediated adhesion mechanism.

Bacterial Adhesion↗
PubMed1992

Enteroaggregative Escherichia coli strains secrete a heat-labile toxin antigenically related to E. coli hemolysin.

A protein toxin of approximately 120,000 Da secreted by nonhemolytic enteroaggregative Escherichia coli strains cross-reacted in Western blots (immunoblots) with antibodies raised against the C-terminal region of E. coli hemolysin. Treatment of HEp-2 cells with enteroaggregative E. coli or culture supernatants caused elevation of intracellular calcium and stimulated calcium-dependent protein phosphorylation.

Antigens, Bacterial↗
PubMed1991

Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain. Phosphorylation induced by enteropathogenic Escherichia coli and phorbol ester.

Previous studies on the mechanism of enteropathogenic Escherichia coli (EPEC) infection have revealed an increase in the phosphorylation state of a number of proteins in human laryngeal HEp-2 cells. The most prominent was an acidic phosphoprotein(s) of Mr 20-21 kDa. The present study reports: (a) a simple method for purification of phosphorylated 20 kDa protein; (b) identification of the 20 kDa phosphoprotein as myosin light chain; and (c) that the phorbol ester, TPA, also increased the phosphorylation of the 20 kDa myosin light chain. In contrast to the effects of EPEC, TPA stimulation resulted in the dissociation of myosin from the cytoskeleton to the cytosol.

Amino Acids↗
PubMed1991

Attaching and effacing enteropathogenic Escherichia coli as a cause of infantile diarrhea in Bangkok.

To identify Escherichia coli that cause infantile diarrhea in Bangkok, Thailand, E. coli isolated in a year-long study of infantile diarrhea were examined for O and H serotypes and virulence determinants. Classic enteropathogenic E. coli (EPEC) were isolated from 28 of 509 infants with diarrhea (cases) and 11 of 509 age-matched controls (P = .009; odds ratio [OR], 2.64). Most of this difference was attributable to EPEC adherence factor (EAF)-positive EPEC that produced an attachment and effacement lesion, as identified in the fluorescence actin staining assay, isolated from 13 cases and 1 control (P = .003; OR, 13.3). EAF-EPEC was isolated from 15 cases and 10 controls (P = .418; OR, 1.52) and EAF+ non-EPEC from 17 cases and 10 controls (P = .242; OR, 1.72). EAF+EPEC that caused an attachment and effacement lesion was found in 3% of children less than 6 months old with diarrhea who were studied in an outpatient clinic in Bangkok in 1988.

Adhesins, Escherichia coli↗
PubMed1991

Screening for enteropathogenic Escherichia coli in infants with diarrhea by the fluorescent-actin staining test.

The attaching effacing (AE) adherence property is now recognized as an important virulence characteristic of enteropathogenic Escherichia coli (EPEC). The fluorescent-actin staining (FAS) test (S. Knutton, T. Baldwin, P. H. Williams, and A. S. McNeish, Infect. Immun. 57:1290-1298, 1989), which is diagnostic for the AE lesions produced by EPEC (and Vero cytotoxin-producing E. coli), has provided an additional tool with which to investigate this important class of enteric pathogens. In this study, we screened for the AE adherence property in two groups of E. coli isolated from infants with diarrhea by using the FAS test and compared the results with those from O:H serotyping, localized adhesion to HEp-2 cells (LA), and the EPEC adherence factor (EAF) probe. Only 16 of 41 (39%) E. coli strains previously diagnosed as EPEC by O antigen serogrouping were FAS test positive, and of these only 12 belonged to recognized EPEC O:H serotypes; 9 strains which did belong to EPEC O:H serotypes were FAS test negative. Of a second group of 297 untyped E. coli, 7 (2.3%) were FAS test positive, and of these only 2 belonged to EPEC serogroups; 5 belonged to serogroups not regarded as EPEC serogroups or were nontypeable. Of the 23 FAS-test-positive strains identified, 10 were EAF probe positive and showed good LA; 13 were EAF probe negative and showed a quantitatively distinctly poor LA. EAF-positive and EAF-negative strains, however, showed equally good adhesion to human small intestinal mucosa. None of the FAS-test-positive E. coli hybridized with probes for Vero toxins 1 or 2. We conclude that the FAS test is diagnostic not only for classical EPEC and Vero cytotoxin-producing E. coli but also for EPEC strains which are not currently being diagnosed because they belong to serotypes not generally regarded as EPEC serotypes.

Actins↗
PubMed1991

Elevation of intracellular free calcium levels in HEp-2 cells infected with enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) are a class of diarrheagenic organisms that induce a characteristic attaching and effacing lesion in enterocytes and various cultured cell lines. Infection of cultured HEp-2 cells by EPEC isolates 2036-80 (serotype O119) and E2348-69 (serotype O127) resulted in significant elevation of intracellular free calcium levels, determined quantitatively with the fluorescent calcium indicator dye 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8- bis(carboxymethyl)aminoquinoline. This effect, which was not observed on infection with non-lesion-forming E. coli strains, was inhibited by dantrolene, a drug that prevents calcium mobilization from intracellular stores. Moreover, activated protein kinase C in infected cells was dissociated from cell membranes by a process that was inhibited by cyclosporin A, suggesting involvement of the calcium-dependent protease calpain. A qualitative method for observing intracellular calcium fluxes by fluorescence microscopy with the recently described fluorescein-based indicator fluo-3 was used to screen a collection of well-characterized E. coli isolates from patients with infantile enteritis. Increased localized calcium-dependent fluo-3 fluorescence was observed only in HEp-2 cells infected with known lesion-forming EPEC strains. We propose that enhancement of intracellular free calcium levels in enterocytes infected with EPEC would result in formation of the characteristic lesion by calcium-dependent activation of actin-depolymerizing proteins, with eventual loss of absorptive capacity.

Calcium↗