Identification ofEscherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM1-ELISA) procedure

Svennerholm, Ann‐Mari; Holmgren, Jan

doi:10.1007/bf02601701

Cited by 349 publications

(182 citation statements)

References 15 publications

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“…The relative levels of cholera toxin produced by wild type and vcpD mutant strains was assessed by cholera toxin ELISA as previously described (Svennerholm and Holmgren, 1978). All cultures were grown under the appropriate in vitro cholera toxin-inducing conditions defined for each biotype.…”

Section: Cholera Toxin Assaymentioning

confidence: 99%

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Marsh

Taylor

1998

Molecular Microbiology

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SummaryCholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin-like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either El Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the El Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose-sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co-expressed in E. coli. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.

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Section: Cholera Toxin Assaymentioning

confidence: 99%

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Marsh

Taylor

1998

Molecular Microbiology

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“…The B-subunit concentration was determined by a GM1-ELISA, using mAb LT39 as the detecting antibody and a preparation of recombinant CTB of known concentration as a standard (Svennerholm and Holmgren, 1978). For the GM1-affinity experiments, the concentration of each B-subunit was determined using either Bradford's protein assay (Bradford, 1976) (Bio-Rad) with bovine serum albumin (BSA) of known concentrations as a standard, or by measuring the absorbance at 280 nm.…”

Section: Protein Analysesmentioning

confidence: 99%

“…The concentrations of unbound Bsubunits in the supernatants were then determined by a GM1-ELISA (Svennerholm and Holmgren, 1978). To determine the membrane-binding ability of EtxB(G33D), the protein was biotinylated as described above (as were CTB and LTB for comparison) prior to incubation with the membranes, and the remaining non-bound proteins were determined in microtitre wells coated with avidin (Sigma) followed by incubations with Extravidin-HRP and OPD.…”

Section: Binding To Receptors From Rabbit Intestinementioning

confidence: 99%

Structural basis for differential receptor binding of cholera and Escherichia coli heat‐labile toxins: influence of heterologous amino acid substitutions in the cholera B‐subunit

Bäckström

Shahabi

Johansson

et al. 1997

Molecular Microbiology

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SummaryThe closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1-25 region were combined with those in the 75-83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33→Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.

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“…Putative campylobacter colonies were identified by biochemical methods (Kaplan, 1980). The rest of the faecal sample was refrigerated and sent for identification of rotaviruses by electron microscopy (Rodriguez et al 1977) and eggs and cysts of parasites and protozoa by a concentration method (Faust, Russell & Jung, 1970 The same E. coli strains from each faecal culture were tested for production of heat-labile (LT) enterotoxin with a GM1-ELISA test (Svennerholm & Holmgren, 1978), using purified ganglioside kindly provided by Professor Lars Svennerholm, Institute of Neurochemistry, Gothenburg, Sweden, and for heat-stable (ST) enterotoxin with the infant mouse assay (Dean et al 1972).…”

Section: Diarrhoeal Disease In Mexican Childrenmentioning

confidence: 99%

Prospective study of diarrhoeal disease in a cohort of rural Mexican children: incidence and isolated pathogens during the first two years of life

Cravioto¹,

Reyes²,

Ortega³

et al. 1988

Epidemiol. Infect.

137

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SUMMARYColonization of the intestine by putative pathogens was followed longitudinally in a cohort of 56 infants born during one calendar year in a rural Mexican village with faecal cultures taken every fortnight and every time a child had diarrhoea. The frequency of isolation of pathogens during episodes of diarrhoea was compared with that of matched controls from the same cohort. Incidence of diarrhoea during the first year of life was 98%, diminishing to 93% during the second year. The incidence curves for each year were not significantly different (P > 041). Isolation of enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli producing heat-stable (ST) and/or heat-labile (LT) enterotoxins and rotaviruses was significantly higher in infants with diarrhoea during the first 2 years of life. In the case of shigella, although no significant differences were found by semester of life, 13 of 16 children in which these strains were found had diarrhoea. Isolation of Salmonella spp., Campylobacter spp. and protozoa were not significantly different in the two groups during the period studied. Strains showing localized adherence to HEp-2 cells or the presence of colonization factor antigens I or E8775 were found with significantly higher frequency in children with diarrhoea. Eighty-two percent of ST' or LT' ETEC strains isolated produced one of the three known colonization factors.

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Identification ofEscherichia coli heat-labile enterotoxin by means of a ganglioside immunosorbent assay (GM1-ELISA) procedure

Cited by 349 publications

References 15 publications

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

Structural basis for differential receptor binding of cholera and Escherichia coli heat‐labile toxins: influence of heterologous amino acid substitutions in the cholera B‐subunit

Prospective study of diarrhoeal disease in a cohort of rural Mexican children: incidence and isolated pathogens during the first two years of life

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