Adherence of Streptococcus pneumoniae to respiratory epithelial cells is inhibited by sialylated oligosaccharides

Abstract

To study carbohydrate-mediated adherence of Streptococcus pneumoniae to the human airway, we measured binding of live S. 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. pneumoniae capsular types studied was inhibited preferentially by sialylated oligosaccharides that terminate with the disaccharide NeuAc alpha2-3(or 6)Galbeta1. Adherence of some strains also was weakly inhibited by oligosaccharides that terminate with lactosamine (Galbeta1-4GlcNAcbeta1). 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 NeuAc alpha2-3(or 6)Galbeta1 is arranged on the bacterial surface in such a manner that it may be cross-linked by oligosaccharides covalently linked to human serum albumin.

FIG. 1

Growth phase-dependent adherence of S. pneumoniae to Chang conjunctival cells. At hourly intervals growth of S. pneumoniae R-6 labeled with [³H]lysine in suspension culture was monitored by light scattering (A₅₉₅) (▵) and aliquots of bacteria were withdrawn, washed, and resuspended in L-15–BSA or L-15–BSA plus 6 mM 6′SLn (□), 6 mM LNnT (▿), or 6 mM GalNAcβ1-3LacNAc (○); they were then added to flat-bottom microtiter wells in which Chang cells had been previously grown to confluence. The fraction of bacteria adherent to cells in the presence of each oligosaccharide inhibitor was determined by scintillation counting and plotted as the percent decrease in binding with respect to the control. Each point represents the mean of at least three determinations in one experiment; for visual clarity, the error was not plotted.

FIG. 2

Inhibition of adherence of S. pneumoniae R-6 to NHBE cells by oligosaccharides. S. pneumoniae R-6 sampled from Columbia broth suspension culture at mid-exponential phase was washed in L-15–BSA and suspended at a density of 10⁹ organisms per ml in the same buffer alone (a) or containing 5 mM 6′SLNnT (b), 5 mM 3′SLNnT (c), or 5 mM LNnT (d); bacteria were then incubated with NHBE cell monolayers for 30 min at room temperature. The slides were then washed, fixed, stained with Giemsa stain, and photographed at a magnification of ×800 under bright-field illumination.

FIG. 3

Inhibition of binding of S. pneumoniae R-6 at mid-exponential phase to Chang conjunctival cells by oligosaccharides. S. pneumoniae R-6 sampled from tryptic soy broth suspension culture at mid-exponential phase was washed in L-15–BSA and resuspended in the same buffer containing 3′SL-HSA (▾), 6′SL-HSA (▴), 3′SL (▿), 6′SL (▵), or 6′SLn (○) at various concentrations; the bacteria were then incubated with Chang conjunctival cells previously grown to confluence in microtiter wells. After incubation for 30 min at room temperature, the wells were washed and counted by liquid scintillation. The fraction of bacteria adherent to cells in the presence of each oligosaccharide inhibitor is plotted as a percentage of binding observed in the absence of inhibitor at each time point. HSA alone, at concentrations comparable to those used for the glycoconjugates, did not inhibit the adherence of S. pneumoniae to Chang cells (data not shown).