Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes
Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes
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DOI：
10.1128/iai.65.10.4199-4206.1997
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发表时间：
1997-10-01
影响因子：
3.1
通讯作者：
Elbein, AD
中科院分区：
文献类型：
--
作者：
Pan, YT;Xu, B;Elbein, AD
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 Man(9)(GlcNAc)(2)-tyrosinamide, which was considerably better than other tyrosinamide-linked oligosaccharides such as Man(7)(GlcNAc)(2), Man(6)(GlcNAc)(2) or Man(5)(GlcNAc)(2). Further evidence that the bacteria preferred Man,(GlcNAc), 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 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-mannose structures was purified from isolated pill. 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. Pill were labeled 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 fewer mannose residues. Polyclonal antibody against this 35-kDa protein also 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 toward Man(9)(GlcNAc)(2) oligosaccharides.
