Helicobacter pylori, the ulcer pathogen residing in the human stomach, binds to epithelial cells of the gastric antrum. We have examined binding of 13 bacterial isolates to epithelial cell lines by use of a sensitive microtiter plate method in which measurement of bacterial urease activity provides the means for quantitation of bound organisms. Several established human gastrointestinal carcinoma cell lines grown as monolayers were compared for suitability in these assays, and the duodenum-derived cell line HuTu-80 was selected for testing bacterial binding inhibitors. When bacteria are pretreated with oligosaccharides, glycoproteins, and glycolipids, a complex picture of bacterial-epithelial adherence specificities emerges. Among the monovalent inhibitors tested, 3-sialyllactose (NeuAc␣2-3Gal␤1-4Glc; 3SL) was the most active oligosaccharide, inhibiting adherence for recent clinical isolates of H. pylori with a millimolar 50% inhibitory concentration (IC 50). Its ␣2-6 isomer (6SL) was less active. Most of the recent clinical isolates examined were inhibited by sialyllactose, whereas long-passaged isolates were insensitive. Among the long-passaged bacterial strains whose binding was not inhibited by 3SL was the strain ATCC 43504, also known as NCTC 11637 and CCUG 17874, in which the proposed sialyllactose adhesin was recently reported to lack surface expression (

To study carbohydrate-mediated adherence of Streptococcus pneumoniae to the human airway, we measured binding of liveS. pneumoniae organisms to a cultured cell line derived from the lining of the conjunctiva and to primary monolayers of human bronchial epithelial cells in the presence and absence of oligosaccharide inhibitors. Both encapsulated and nonencapsulated strains of S. pneumoniae grown to mid-logarithmic phase in suspension culture adhered to cultured primary respiratory epithelial cells and the conjunctival cell line. Adherence of nine clinically prevalent S. pneumoniaecapsular types studied was inhibited preferentially by sialylated oligosaccharides that terminate with the disaccharide NeuAcα2-3(or 6)Galβ1. Adherence of some strains also was weakly inhibited by oligosaccharides that terminate with lactosamine (Galβ1-4GlcNAcβ1). When sialylated oligosaccharides were covalently coupled to human serum albumin at a density of approximately 20 oligosaccharides per molecule of protein, the molar inhibitory potency of the oligosaccharide inhibitor was enhanced 500-fold. The above-mentioned experiments reveal a previously unreported dependence upon sialylated carbohydrate ligands for adherence of S. pneumoniae to human upper airway epithelial cells. Enhanced inhibitory potencies of polyvalent over monovalent forms of oligosaccharide inhibitors of adherence suggest that the putative adhesin(s) that recognizes the structure NeuAcα2-3(or 6)Galβ1 is arranged on the bacterial surface in such a manner that it may be cross-linked by oligosaccharides covalently linked to human serum albumin.

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.

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