Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain

Dubreuil, J. Daniel; Fairbrother, John M.; Lallier, R.; Larivière, Serge

doi:10.1128/iai.59.1.198-203.1991

Cited by 34 publications

(24 citation statements)

References 23 publications

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“…It has been shown that STII is a poor immunogen. Dubreuil et al (5) injected STII emulsified in complete Freund's adjuvant into rabbits. Inoculations were repeated eight times with incomplete Freund's adjuvant.…”

Section: Discussionmentioning

confidence: 99%

Some properties of purified Escherichia coli heat-stable enterotoxin II

et al. 1992

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We examined the biological properties of purified Escherichia coli heat-stable enterotoxin H (STH) using mouse intestinal loop assays and compared these properties with those of heat-stable enterotoxin I (STI) and cholera toxin (CT). The action of STU over time differed from those of STI and CT. STH did not alter cyclic GMP or cyclic AMP levels in intestinal mucosal cells. Our results supported the idea that the mechanism of action of STII in inducing fluid secretion is different from the mechanisms of action of STI and CT. This hypothesis was further supported by the fact that an anti-STII neutralizing serum did not neutralize the activities of STI and CT. Subsequently, we examined the involvement of prostaglandins in the action of STlI. The level of prostaglandin E2 in the fluid accumulated as a result of the action of STU increased, and the prostaglandin synthesis inhibitors aspirin and indomethacin significantly reduced the response to STH. These results implicate prostaglandin E2 in the mechanism of action of STH.

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Section: Discussionmentioning

confidence: 99%

Some properties of purified Escherichia coli heat-stable enterotoxin II

et al. 1992

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“…Horseradish peroxidase‐conjugated goat anti‐rabbit antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (Mississauga, ON, Canada). Polyclonal antibodies to STb toxin were produced by immunizing rabbits with pure STb toxin [20]. High performance thin‐layer chromatography (HP‐TLC) plates were purchased from E. Merck (EM Science, Gibbstown, Germany).…”

Section: Methodsmentioning

confidence: 99%

LC-MS analysis of pig intestine sulfatides: Interaction withEscherichia coliSTb enterotoxin and characterization of molecular species present

Beausoleil

Lépine

Dubreuil

2002

FEMS Microbiology Letters

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STb, a 48-amino acid thermostable enterotoxin is produced by enterotoxigenic Escherichia coli strains and is responsible for diarrheal diseases in many animals, including man. Our laboratory recently identified a family of molecules, from a lipid extract of porcine intestinal epithelial cells, that could bind to STb. These molecules were identified as sulfatides as they reacted with a monoclonal antibody raised against this family of molecules. However, as the epitope recognized by this monoclonal antibody was the galactose 3-sulfate, a doubt could remain as to the exact nature of the identified receptors. The goal of this study was thus to confirm the chemical nature of the STb-binding molecule as sulfatides or as distinctive molecules comprising a sulfated galactosyl residue. Using a thin-layer chromatography-overlay method we confirmed using antibodies to STb that STb recognizes the commercial sulfatides and a band migrating at the same level from the intestinal tissue lipid extract obtained from an 8-week-old piglet. The compounds recovered from the silica gel plates were analyzed by mass spectrometry in electrospray negative-ionization mode. The most abundant ions observed had m/z values of 779, 795, 879 and 907. For commercial bovine brain sulfatides the ions 795, 879 and 907 have been attributed to hydroxylated sulfatides with a saturated fatty acid chain containing 16, 22 and 24 carbons, while the 779 ion contained a saturated fatty acid chain of 16 carbons. The general profile of the ions observed was similar to the already described commercial bovine brain sulfatides.

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“…Alkaline phosphatase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse Ig were obtained from Jackson ImmunoResearch Laboratories, Inc. (Mississauga, Ontario, Canada). Polyclonal antibodies to STb toxin were produced by immunizing rabbits with pure STb (12). Mouse monoclonal antibody to sulfatide was kindly provided by Pam Fredman, University of Göteborg, Göteborg, Sweden.…”

Section: Methodsmentioning

confidence: 99%

“…The purity of the toxin was routinely verified by N-terminal sequence analysis using Edman degradation (Applied Biosystem model 470A gas-phase sequencer) as described before (18). The biological activity of STb was assessed by the rat ligated intestinal loop assay (12).…”

Section: Methodsmentioning

confidence: 99%

Sulfatide from the Pig Jejunum Brush Border Epithelial Cell Surface Is Involved in Binding of Escherichia coli Enterotoxin b

1998

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Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3′-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)β1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277–288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.

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Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain

Cited by 34 publications

References 23 publications

Some properties of purified Escherichia coli heat-stable enterotoxin II

Some properties of purified Escherichia coli heat-stable enterotoxin II

LC-MS analysis of pig intestine sulfatides: Interaction withEscherichia coliSTb enterotoxin and characterization of molecular species present

Sulfatide from the Pig Jejunum Brush Border Epithelial Cell Surface Is Involved in Binding of Escherichia coli Enterotoxin b

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