title: Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes.
abstract: Enterobacter cloacae has been implicated as one of the causative agents in neonatal infection and causes a septicemia thought to be initiated via the gastrointestinal tract. The adhesion of radiolabeled E. cloacae to HT-29 cells was concentration and temperature dependent and was effectively blocked by unlabeled bacteria or by millimolar concentrations of alpha-mannosides and micromolar concentrations of high-mannose oligosaccharides. A variety of well-characterized mannose oligosaccharides were tested as inhibitors of adhesion. The best inhibitor was the Man9(GlcNAc)2-tyrosinamide, which was considerably better than other tyrosinamide-linked oligosaccharides such as Man7(GlcNAc)2, Man6(GlcNAc)2 or Man5(GlcNAc)2. Further evidence that the bacteria preferred Man9(GlcNAc)2 structures was obtained by growing HT-29 cells in the presence of glycoprotein processing inhibitors that block mannosidase I and increase the amount of protein-bound Man9(GlcNAc)2 at the cell surface. Such cells bound 1.5- to 2-fold more bacteria than did control cells. The adhesin involved in binding to high-mannose structures was purified from isolated pili. On sodium dodecyl sulfate-gels, a 35-kDa protein was identified by its specific binding to a mannose-containing biotinylated albumin. The amino acid sequences of several peptides from the 35-kDa subunit showed over 85% identity to FimH, the mannose-specific adhesin of Salmonella typhimurium. Pili were labeled with 125I and examined for the ability to bind to HT-29 cells. Binding showed saturation kinetics and was inhibited by the addition of Man9(GlcNAc)2-tyrosinamide but not by oligosaccharides with fewer mannose residues. Polyclonal antibody against this 35-kDa protein also effectively blocked adhesion of pili or E. cloacae, but no effect was observed with nonspecific antibody. These studies demonstrate that the 35-kDa pilus subunit is a lectin whose specificity is directed toward Man, (GlcNAc)2 oligosaccharides.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
authors.forename: Y T
authors.initials: YT
authors.lastname: Pan
authors.forename: B
authors.initials: B
authors.lastname: Xu
authors.forename: K
authors.initials: K
authors.lastname: Rice
authors.forename: S
authors.initials: S
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Jackson
authors.forename: A D
authors.initials: AD
authors.lastname: Elbein
mesh_terms: D017485:1-Deoxynojirimycin / Q000494:pharmacology; D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018830:Adhesins, Escherichia coli; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion / Q000502:physiology*; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D016972:Enterobacter cloacae / Q000472:pathogenicity*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000737:chemistry / Q000378:metabolism*; D007212:Indolizines / Q000494:pharmacology; D037102:Lectins / Q000378:metabolism; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D017026:Swainsonine / Q000494:pharmacology; D054836:Indolizidines
chemical_list: D017485:1-Deoxynojirimycin; D018829:Adhesins, Bacterial; D018830:Adhesins, Escherichia coli; D000470:Alkaloids; D036781:Fimbriae Proteins; D007212:Indolizines; D037102:Lectins; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D009844:Oligosaccharides; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D017026:Swainsonine; C058769:fimH protein, E coli; C065629:kifunensine; C037806:castanospermine; D054836:Indolizidines