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S Knutton

Publications and source records attributed to S Knutton.

At least 37 records · Page 2Linked to original sources
PubMed1998

Enteropathogenic Escherichia coli virulence genes encoding secreted signalling proteins are essential for modulation of Caco-2 cell electrolyte transport.

The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhea remains uncertain. In vitro, EPEC stimulates a rapid increase in short-circuit current (Isc) across Caco-2 cell monolayers coincident with intimate attaching and effacing (A/E) bacterial adhesion. This study has examined the roles of specific EPEC virulence proteins in this Isc response. EPEC genes encoding EspA, EspB, and EspD, essential for signal transduction in host cells and A/E activity, were also required for modulation of Caco-2 electrolyte transport.

Adhesins, Bacterial↗
PubMed1998

Increased levels of intracellular calcium are not required for the formation of attaching and effacing lesions by enteropathogenic and enterohemorrhagic Escherichia coli.

Elevated concentrations of intracellular calcium ([Ca]i) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic Escherichia coli (EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]i in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]i at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic E. coli (EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]i in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]i are not required for A/E lesion formation by EPEC and EHEC.

Calcium↗
PubMed1998

Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens.

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.

Adhesins, Bacterial↗
PubMed1998

Rapid modulation of electrolyte transport in Caco-2 cell monolayers by enteropathogenic Escherichia coli (EPEC) infection.

BACKGROUND AND AIMS: The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhoea remains uncertain. EPEC adhere to enterocytes and transduce signals which produce a characteristic "attaching and effacing" (A/E) lesion in the brush border membrane. The present in vitro study was designed to determine whether signal transduction by EPEC also influences electrolyte transport. METHODS: Caco-2 cell monolayers were rapidly infected with wild type EPEC strain E2348/69, or the signal transduction-defective mutant 14.2.1(1), and mounted in Ussing chambers. RESULTS: Strain E2348/69 stimulated a rapid but transient increase in short circuit current (Isc) which coincided with A/E lesion formation; this Isc response was absent on infection with strain 14.2.1(1). While the initial rise in Isc induced by E2348/69 was partially (approximately 35%) dependent on chloride, the remainder possibly represents an influx of sodium and amino acid(s) across the apical membrane. CONCLUSIONS: The study directly shows that, after initial adhesion, EPEC induce major alterations in host cell electrolyte transport. The observed Isc responses indicate a rapid modulation of electrolyte transport in Caco-2 cells by EPEC, including stimulation of chloride secretion, for which signal transduction to host cells is a prerequisite.

Amino Acids↗
PubMed1997

Down regulation of intimin expression during attaching and effacing enteropathogenic Escherichia coli adhesion.

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions in the intestinal mucosa. The intimate bacterial adhesion associated with A/E lesion formation is promoted by intimin, a 94-kDa EPEC surface protein. Anti-intimin antisera raised in rabbits by using the purified 280-amino-acid cell binding domain of intimin as the immunogen were employed in immunofluorescence and immunoelectron microscopical studies to investigate the expression of intimin by classical EPEC strain E2348/69 (O127:H6) and defined E2348/69 derivatives during culture growth and A/E bacterium adhesion to cultured HEp-2 cells. In stationary-phase broth cultures, only a small fraction of E2348/69 bacteria expressed intimin, and of those that did, immunolabelling revealed a uniform distribution of intimin over the bacterial surface; increased numbers of bacteria expressing intimin were detected when E2348/69 was grown in tissue culture medium, an effect not seen with strain JPN15, a virulence plasmid-cured derivative of E2348/69. Strain CVD206, an eaeA mutant of E2348/69, did not stain with the anti-intimin antisera, but strain CVD206(pCVD438), containing a functional eaeA gene, stained uniformly. After a 3-h incubation of HEp-2 cells with strain E2348/69, double immunofluorescence labelling of intimin and cellular actin revealed strong intimin expression by all A/E bacteria, but after 6 h of incubation, intimin expression by most E2348/69 bacteria was greatly reduced or not detected. This effect on intimin expression was not observed with strain JPN15 but was restored for strain JPN15(pCVD450) harboring the virulence plasmid-encoded per genes. These results indicate that surface expression of intimin is regulated by environmental factors during bacterial growth and following A/E lesion formation and that virulence plasmid-encoded genes participate in these regulation processes.

Actins↗
PubMed1996

Phosphorylation of myosin light chain at distinct sites and its association with the cytoskeleton during enteropathogenic Escherichia coli infection.

Myosin light chain, the most prominent host cell phosphoprotein during adhesion of enteropathogenic Escherichia coli to cultured HEp-2 cells, was shown to be distributed between cytosolic and cytoskeletal cell fractions; its association with the cytoskeletal fraction increased with time of enteropathogenic E. coli incubation. Phosphopeptide mapping indicated cytosolic and cytoskeletal myosin light chain phosphorylation at different sites by protein kinase C and myosin light chain kinase.

Cells, Cultured↗
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↗
PubMed1995

Cellular responses to enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC), first described in the 1940's and 1950's, remain an important cause of severe infantile diarrhoea in many parts of the developing world. EPEC do not produce enterotoxins and are not invasive; instead their virulence depends upon exploitation of host cell signalling pathways and the host cell cytoskeleton both as a means of colonizing mucosal surfaces of the small intestine and causing diarrhoea. Following initial mucosal attachment, EPEC secrete 'signalling' proteins and express a surface adhesin, intimin, to produce 'attaching & effacing' lesions in the enterocyte brush border membrane characterised by localised destruction of brush border microvilli, intimate bacterial adhesion and cytoskeletal reorganisation and accretion beneath attached bacteria. The pathophysiology of EPEC diarrhoea is also complex and probably results from a combination of epithelial cell responses including both electrolyte secretion and structural damage.

Bacterial Adhesion↗
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↗