Role of the putative 'link' glycopeptide of intestinal mucin in binding of piliated Escherichia coli serotype O157:H7 strain CL-49

Abstract

Purified rat intestinal mucin was used to identify mucin-binding sites for type 1-piliated Escherichia coli O157:H7 strain CL-49 isolated from a patient with hemorrhagic colitis and hemolytic uremic syndrome. Optimum binding of bacteria in a microtiter binding assay occurred with a mucin coating concentration of 15 μg (protein)/150 μI. In hapten inhibition studies, several nonmucin glycoproteins bearing exposed mannosyl residues in N-linked oligosaccharides were effective inhibitors, as was rat mucin. The same glycoproteins caused bacterial aggregation. High-molecular-mass glycoproteins of the mucin were separated from its 118-kilodalton 'link' glycopeptide fraction, and the latter was shown to be the mucin-binding component for E. coli CL-49 and its purified type 1 pili. This was confirmed in hemagglutination inhibition studies. Treatment of the link glycopeptide with jack bean α-mannosidase or endo-β-N-acetylglucosaminidase H destroyed bacterial binding activity. Chemical or enzymatic modifications of intact rat mucin were undertaken to evaluate the normal accessibility of the link glycopeptide receptors to E. coli CL-49. Deglycosylation with trifluoromethane-sulfonic acid abolished binding, whereas pronase digestion had no effect. Reduction and alkylation as well as lipid extraction enhanced bacterial binding by the mucin, presumably by causing greater exposure of receptor sites. In summary, our binding studies revealed, for the first time, that intestinal mucin bears oligomannosyl receptors for type 1 pili and that these receptors are located on N-linked oligosaccharides of the 118-kilodalton link glycopeptide region of the mucin. Our experiments suggest the receptors are normally partly 'covered' by noncovalently bound lipid. In addition, release of the link component from the rest of the mucin by disulfide bond reduction causes greater exposure of specific bacterium-binding sites.