Search PubMed⌕ Search

Biomedical subjects

S Knutton

Publications and source records attributed to S Knutton.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1990

Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli.

Infection of HEp-2 monolayers with enteropathogenic Escherichia coli 2036-80 (O119) stimulated phosphorylation of several target cell proteins, the most prominent of which had apparent molecular weights of 21,000 and 29,000. Proteins of the same size were phosphorylated in response to known activators of the calcium-phospholipid-dependent protein kinase C. Screening of clinical isolates of various O serogroups revealed that all strains able to form the characteristic attaching and effacing lesion of enteropathogenic E. coli showed elevated phosphorylation of 21,000- and 29,000-dalton protein species.

Bacterial Adhesion↗
PubMed1989

Adhesion and ultrastructural properties of human enterotoxigenic Escherichia coli producing colonization factor antigens III and IV.

Human enterotoxigenic Escherichia coli (ETEC) producing colonization factor antigen III (CFA/III) and coli surface antigens 4, 5, and 6 (CS4, CS5, and CS6) of CFA/IV were examined ultrastructurally and for ability to adhere to human small intestinal enterocytes and to cultured human intestinal mucosa. Strains of serotypes O25:H-, O25:H42, and O167:H5 producing CFA/III plus CS6, CS4 plus CS6, and CS5 plus CS6, respectively, showed good adhesion to human enterocytes (1.8 to 4.2 bacteria per brush border) and cultured human intestinal mucosa, whereas variants lacking these antigens or producing only CS6 were nonadherent (0 to 0.03 bacterium per brush border). By electron microscopy, CFA/III, CS4, and CS5 appeared as morphologically distinct rodlike fimbriae: CFA/III was 7 to 8 nm in diameter, CS4 was 6 to 7 nm in diameter, and CS5 was 5 to 6 nm in diameter. CS5 was unusual in that it appeared to be composed of two fine fibrils arranged in a double-helical structure. CS6 was difficult to characterize morphologically but possibly has a very fine fibrillar structure. By specific fimbrial staining and immunoelectron microscopy. CS4 and CS5 were shown to promote mucosal adhesion of ETEC; a similar adhesion role for the CS6 antigen could not be confirmed. ETEC strains of serotypes O27:H7, O27:H20, O148:H28, and O159:H20 which produced CS6 showed good adhesion to human enterocytes (1.6 to 3.0 bacteria per brush border), whereas variants which lacked CS6 were nonadherent (0 to 0.01 bacterium per brush border). These strains, however, also produced fimbrial or fibrillar surface antigens, in addition to CS6, which probably represent additional coli surface antigens responsible for the observed adhesive properties of these ETEC serotypes.

Antigens, Bacterial↗
PubMed1989

Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) adhere to the intestinal mucosa and produce an attaching and effacing (AE) lesion in the brush border microvillous membrane; the AE lesion is characterized by localized destruction of microvilli and intimate attachment of bacteria to the apical enterocyte membrane. A similar lesion is seen when bacteria adhere in vitro to a variety of human tissue culture cell lines. In both cases, dense concentrations of microfilaments are present in the apical cytoplasm beneath attached bacteria. Using a fluorescein-labeled phallotoxin, we have shown that these microfilaments are composed of actin. Cells infected with EPEC and EHEC strains known from electron microscopic studies to produce the AE lesion all exhibited intense spots of fluorescence which corresponded in size and position with each adherent bacterium; cells infected with adherent E. coli strains known not to produce the AE lesion did not produce this striking pattern of fluorescence and were indistinguishable from uninfected control cells. These results indicate that such site-specific concentrations of cytoskeletal actin are characteristic of the AE membrane lesion and can form the basis of a simple, highly sensitive diagnostic test for EPEC and EHEC.

Actins↗
PubMed1987

Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa.

The adhesion of classic enteropathogenic Escherichia coli (EPEC) strains of human origin to isolated human small intestinal enterocytes and cultured small intestinal mucosa was investigated. An adhesion assay with isolated human enterocytes prepared from duodenal biopsy samples was developed and tested with EPEC strains known to cause diarrhea in healthy adult volunteers. In the assay a mean of 53 and 55% of enterocytes had brush border-adherent E. coli E2348 (O127;H6) and E851 (O142:H6), respectively, whereas the value for a nonpathogenic control strain and a plasmid-cured derivative of strain E2348 was 0%. A collection of 17 EPEC strains was also tested for the ability to colonize cultured human duodenal mucosa. Extensive colonization occurred with 13 strains, including serogroups O55, O86, O111, O114, O119, O127, O128, and O142; and in each case electron microscopic examination of colonized mucosa revealed the characteristic histopathological lesion reported by others in natural and experimental EPEC infections. EPEC strains were seen to adhere intimately to the enterocyte surface, causing localized destruction of microvilli. The plasmid-cured derivative of strain E2348, which colonized cultured mucosa much less efficiently than the parent strain, nevertheless produced an identical lesion, indicating that plasmid-encoded factors are not essential for adhesion and the brush border-damaging property of EPEC.

Bacterial Adhesion↗
PubMed1987

Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells.

Plasmid-encoded adherence factors have been shown to be important for the full expression of enteropathogenic Escherichia coli (EPEC) pathogenicity and for EPEC adhesion to cultured HEp-2 cells. EPEC strain E2348 (O127) shows localized HEp-2 cell adhesion and possesses a 60-megadalton plasmid, pMAR2. When E2348 is cured of pMAR2 it loses the ability to adhere to HEp-2 cells, while nonadherent E. coli K-12 strains P678-54 and HB101 acquire HEp-2 adhesiveness after they gain the plasmid. By electron microscopy, E2348 was seen to adhere to HEp-2 cells in a manner that closely resembled EPEC adhesion to intestinal mucosa; bacteria were intimately attached to projections of the apical HEp-2 cell membrane and caused localized destruction of microvilli. The plasmid-containing K-12 strains, on the other hand, did not show intimate attachment and there was no modification of cell surface architecture. It is concluded that plasmid pMAR2 codes for an adhesin, possibly fimbrial in nature, that promotes HEp-2 adhesion but that other chromosomally encoded factors are required for EPEC to achieve the characteristic mode of intimate cell attachment.

Bacterial Adhesion↗
PubMed1987

Identification of a new fimbrial structure in enterotoxigenic Escherichia coli (ETEC) serotype O148:H28 which adheres to human intestinal mucosa: a potentially new human ETEC colonization factor.

Three important fimbrial colonization factor antigens (CFAs) designated CFA/I, CFA/II, and E8775 were identified originally in some human enterotoxigenic Escherichia coli (ETEC) strains because of their mannose-resistant hemagglutination properties. To identify CFA, in strains lacking mannose-resistant hemagglutination properties we exploited the ability of human ETEC strains to adhere to human proximal small intestinal mucosa. ETEC strain B7A (O148:H28) was selected for study because it belongs to an epidemiologically important serotype and does not produce a known CFA, and yet it is known to be pathogenic and cause diarrheal disease in human volunteers. Results of an human enterocyte adhesion assay indicated that some bacteria in cultures of B7A produced adhesive factors. To select for such bacteria, cultured human duodenal mucosal biopsy samples were infected with B7A for up to 12 h, after which time a large percentage of the mucosal surface became colonized by bacteria. A new fimbrial structure morphologically distinct from CFA/I, CFA/II, and E8775 fimbriae and consisting of curly fibrils (approximately 3 nm in diameter) was readily identified when bacteria were subcultured from the mucosa and examined by electron microscopy. Identical fimbriae were produced by ETEC strain 1782-77 of the same serotype. Identification of these fimbriae only on bacteria subcultured from human intestinal mucosa strongly suggests that they promote mucosal adhesion of ETEC serotype O148:H28 and thus represent a potentially new human ETEC CFA.

Bacterial Adhesion↗
PubMed1987

Adherence to human colonocytes of an Escherichia coli strain isolated from severe infantile enteritis: molecular and ultrastructural studies of a fibrillar adhesin.

Escherichia coli 469-3 (O21:H-) was isolated from a child with severe enteritis. Ultrastructural analysis of the surface of the strain indicated the presence of very fine fimbriae which mediated mannose-resistant hemagglutination of human blood and caused the bacteria to adhere to human epithelial cell lines and to brush borders of isolated human colonic, but not duodenal, enterocytes. A cosmid library of total DNA of the strain, expressed in laboratory strains of E. coli, was screened by a rapid hemadsorption method, and a number of positive clones were identified. Restriction endonuclease fragments specifying mannose-resistant adherence were subcloned from the cosmid DNA of a strongly hemagglutinating clone in a plasmid vector. The identity of the adhesin was confirmed by biochemical, electron-microscopic, and immunological comparisons with the adhesin synthesized by the clinical isolate. It comprised a high-molecular-weight aggregate of a 14,000-dalton subunit protein which bound antiserum raised against the mannose-resistant adhesin of strain 469-3. The adhesin was synthesized by both the clone and the parental strain at growth temperatures above 18 degrees C but by only a fraction of the cells in a pure culture, although all the bacteria which adhered to human cells expressed the protein.

Adhesins, Escherichia coli↗
PubMed1985

Adhesion of enterotoxigenic Escherichia coli to human small intestinal enterocytes.

An improved enterocyte adhesion assay has been used to examine a collection of 44 strains of enterotoxigenic Escherichia coli (ETEC) for their ability to adhere to the brush border of isolated human duodenal enterocytes. Fourteen strains showed good adhesion; in each case the ability to adhere correlated with the production of colonization factor antigen I or II (CFA/I or CFA/II) fimbriae. CFA/II-positive producing coli surface antigens 1 and 3 (CS1 and CS3), coli surface antigens 2 and 3 (CS2 and CS3), and only coli surface antigen 3 (CS3) each showed good adhesion. CS3-mediated brush border attachment of CFA/II-positive ETEC was demonstrated by electron microscopy with monospecific antibody and an immunogold labeling technique. One CFA/I-positive ETEC strain was nonadherent in the assay, as were ETEC producing type 1 somatic fimbriae. Five animal ETEC strains producing K88, K99, F41, and 987P fimbriae were slightly more adhesive than control strains, but adhesion was significantly less than that of CFA-positive ETEC. Twenty five human ETEC strains that lacked CFA/I and CFA/II were nonadherent, suggesting either that the surface antigens responsible for adhesion to human intestinal mucosa in these strains were not being produced or that mucosal receptors for these strains are present in regions of the small intestine other than the duodenum.

Adhesiveness↗