Binding characteristics of Escherichia coli enterotoxin b (STb) to the pig jejunum and partial characterization of the molecule involved.
Escherichia coli heat-stable enterotoxin b (STb) causes severe diarrhoea in weaning piglets. STb most probably has to bind to intestinal epithelial cells in order to achieve its effect. Using biotinylated biologically active STb, we developed a semi-quantitative binding assay using indirect fluorescence microscopy. We demonstrated the attachment of the biotinylated toxin to microvilli of the pig jejunum. However, binding was abolished when biotinylated STb was either boiled or treated with 2-mercaptoethanol, treatments known to abolish biological activity. Different characteristics of STb attachment to the pig small intestine were determined. The reaction was rapid and reached maximum intensity after approximately 10 min. The binding was pH dependent showing an optimum at pH 5.8. Incubation at either 4 degrees C, 25 degrees C or 37 degrees C did not affect the binding. No competition was observed with non-biotinylated STb. However, preincubation of biotinylated STb with streptavidin conjugated to horseradish peroxidase completely abolished the binding. Pig tissues other than jejunum demonstrated binding towards STb including duodenum, ileum, caecum, colon, liver, lung, spleen and kidney. The molecule involved was then partially characterized. Metaperiodate treatment of the jejunum sections abrogated binding but protease treatment had no effect. Enzymatic treatments of jejunal sections demonstrated that N- and O-glycosidases, and several exoglycosidases did not affect binding, whereas reduced binding was observed with ceramide glycanase and alpha-glucosidase, and was completely abolished following neuraminidase treatment. Overall, our results suggest that in vitro STb binding was rapid, pH dependent, temperature independent, not restricted to jejunum and involves a molecule that seems to be composed of a ceramide moiety, terminal neuraminic acid and/or alpha-linked terminal glucose residue(s).
Rousset
E
E
Groupe de recherche sur les malades infectieuses du porc (GREMIP), Département de pathologie et microbiologie, Faculté de médecine vétérinaire, Université de Montréal, C. P. 5000, Saint-Hyacinthe, Québec, J2S 7C6, Canada.
Harel
J
J
Dubreuil
J D
JD
D000818:Animals; D001667:Binding, Competitive; D004064:Digestive System / Q000737:chemistry; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007583:Jejunum / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008856:Microscopy, Fluorescence; D011956:Receptors, Cell Surface / Q000032:analysis*; D013154:Spleen / Q000737:chemistry; D013552:Swine; D013696:Temperature
D004768:Enterotoxins; D011956:Receptors, Cell Surface