ABSTRACT
Enteropathogenic Escherichia coli strains of diffused adherent (DA) and localised adherent (LA) phenotypes were tested for their ability to bind to glycolipids. DA strains did not bind to the glycolipids tested, while LA strains bound to asialo GM1, asialo GM2, globoside and lacto-N-neotetraose in decreasing order of avidity. The minimum common sequence among the four glycolipids could be delineated as GalNac beta 1-4 Gal as the binding epitope with GalNac beta 1-3 Gal and GlcNac beta 1-3 Gal serving as relatively weaker binders. The binding was not inhibited by a variety of free oligosaccharides or by the neoglycoproteins tested. Adhesion-negative mutants of an enteropathogenic LA strain showed a markedly reduced binding to asialo GM1 indicating that the recognition of GalNac beta 1-4 Gal was correlated with the ability to adhere to HeLa cells. Thus recognition and binding to glycolipids could play an important role in colonisation through adherence to intestinal surfaces.
Subject(s)
Carbohydrates/chemistry , Escherichia coli/metabolism , Gangliosides/metabolism , Glycolipids/metabolism , Binding Sites , Carbohydrate Metabolism , Carbohydrate Sequence , Escherichia coli/pathogenicity , G(M1) Ganglioside/chemistry , G(M1) Ganglioside/metabolism , Gangliosides/chemistry , Glycolipids/chemistry , Glycoproteins/pharmacology , HeLa Cells , Humans , Molecular Sequence Data , Oligosaccharides/pharmacology , PhenotypeABSTRACT
A gene (st) coding for heat-stable toxin (STh) was identified from a plasmid of a locally isolated enterotoxigenic Escherichia coli strain. The gene was cloned and its nucleotide (nt) sequence was determined. Comparison of this nt sequence with that of another st gene reported earlier, showed a single nt substitution within the structural gene for ST. This change resulted in the replacement of proline at position 19 by alanine in the STh of the locally isolated strain. The st gene was hyperexpressed using the phage T7 or the tac promoter vector systems. A 20-fold increase in STh yield was obtained in minimal medium culture supernatants following induction of the T7 promoter. There was no significant accumulation of the precursor peptide within the periplasm of the induced cell, indicating efficient processing under conditions of enhanced transcription of the gene. The yield of STh was monitored using a competitive ELISA, which was found to be a simple and sensitive assay for determining STh concentrations. A rapid and efficient isolation procedure for STh has been developed.
