Infect. Immun., 10 1997, 4199-4206, Vol 65, No. 10
Copyright © 1997, American Society for Microbiology

Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes

YT Pan, B Xu, K Rice, S Smith, R Jackson and AD Elbein
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.

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.

This article has been cited by other articles:

• Xie, B., Zhou, G., Chan, S.-Y., Shapiro, E., Kong, X.-P., Wu, X.-R., Sun, T.-T., Costello, C. E. (2006). Distinct Glycan Structures of Uroplakins Ia and Ib: STRUCTURAL BASIS FOR THE SELECTIVE BINDING OF FimH ADHESIN TO UROPLAKIN Ia. J. Biol. Chem. 281: 14644-14653 [Abstract] [Full Text]