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 0157: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 ,ug (protein)/150 ,ul. 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 a-mannosidase or endo-oI-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.

Mucin isolated from rabbit colon inhibits in vitro binding of Escherichia coli RDEC-1

“…The adherence assay employed in the present study took mucin (31). The link glycopeptide of mucin has been found in advantage of the hydrophobic properties of piliated RDEC-1 the mucin molecule from a number of intestinal sites in a (12).…”

“…A number of complementary constituent of mucin is separated from the native mucin and methods were used to show that the middle-density fractions exposed under these conditions (30). Sajjan and Forstner collected from the ultracentrifugations contain purified mu- (31) used similar conditions to demonstrate that the link cin. In contrast to a previous study with Giardia lamblia, in peptide mediated mucin binding to type 1 pili expressed by which the nonmucin protein fractions of small-intestine E. coli CL-49 of serotype 0157:H7.…”

Growth of Salmonella typhimurium SL5319 and Escherichia coli F-18 in mouse cecal mucus: role of peptides and iron

“…Specific intestine mucosal glycoproteins are known to contain oligosaccharides containing galactose, mannose, and N-acetylglucosamine, e.g. complex type-Nglycosidic glycoproteins [18] and mucin "link" glycopeptide [19]. It will be of interest to determine whether such oligosaccharides can be metabolized by S. typhimurium SL5319.…”

Sulfated di‐, tri‐ and tetraantennary N‐glycans in human Tamm‐Horsfall glycoprotein

“…The biological role of THP remains to be clarified. Several studies have indicated that THP is possibly a urinary inhibitor of the binding of Escherichia coli to epithelial cells of the lower urinary tract and bladder by binding via its carbohydrate moiety to several types of carbohydrate receptors on the fimbriae of E. coli (Parkkinen et al, 1983(Parkkinen et al, , 1988Moch et al, 1987;Sajjan and Forstner, 1990). An additional defence mechanism is possibly present in the upper urinary tract where THP is expressed on the endothelium of the thick ascending limb of the loop of Henle.…”