SPECIFICITY OF THE HIGH-MANNOSE RECOGNITION SITE BETWEEN ENTEROBACTER-CLOACAE PILI ADHESIN AND HT-29 CELL-MEMBRANES

Enterobacter cloacae has been implicated as one of the causative agent s in neonatal infection and causes a septicemia thought to be initiate d via the gastrointestinal tract. The adhesion of radiolabeled E. cloa cae to HT-29 cells was concentration and temperature dependent and was effectively blocked by unlabeled bacteria or by millimolar concentrat ions of alpha-mannosides and micromolar concentrations of high mannose oligosaccharides. A variety of well-characterized mannose oligosaccha rides were tested as inhibitors of adhesion. The best inhibitor was th e Man(9)(GlcNAc)(2)-tyrosinamide, which was considerably better than o ther tyrosinamide-linked oligosaccharides such as Man(7)(GlcNAc)(2), M an(6)(GlcNAc)(2) or Man(5)(GlcNAc)(2). Further evidence that the bacte ria preferred Man,(GlcNAc), structures was obtained by growing HT-29 c ells in the presence of glycoprotein processing inhibitors that block mannosidase I and increase the amount of protein-bound Man(9)(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-mannos e structures was purified from isolated pill. On sodium dodecyl sulfat e-gels, a 35-kDa protein was identified by its specific binding to a m annose-containing biotinylated albumin. The amino acid sequences of se veral peptides from the 35-kDa subunit showed over 85% identity to Fim H, the mannose-specific adhesin of Salmonella typhimurium. Pill were l abeled with I-125 and examined for the ability to bind to HT-29 cells. Binding showed saturation kinetics and was inhibited by the addition of Man(9)(GlcNAc)(2)-tyrosinamide but not by oligosaccharides with few er mannose residues. Polyclonal antibody against this 35-kDa protein a lso effectively blocked adhesion of pill 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 to ward Man(9)(GlcNAc)(2) oligosaccharides.