Characterization of salivary alpha-amylase binding to Streptococcus sanguis

Abstract

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, a-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with _251-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of a-amylase which bound to S. sanguis. Purified oa-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [1251]ot-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled oi-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated a-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch. * Corresponding author. been shown to interact with various oral bacteria in vitro. These include mucins (20,28,37,38), proline-rich glycoprotein (5, 56), secretory immunoglobulin A (sIgA) (40, 64), a fucose-rich glycoprotein from parotid saliva (16), lysozyme (25, 52), P2-microglobulin (15), and others (3,4). The goal of the present study was to ascertain to what extent salivary components interact with S. sanguis and other bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. MATERIALS AND METHODSMaterials. The following materials were obtained from the indicated sources: Trypticase soy broth (TSB) and agar (BBL Microbiology Systems, Cockeysville, Md.); yeast extract (Difco Laboratories, Detroit, Mich.); crude human salivary a-amylase, Bacillus amyloliquefaciens ot-amylase, Aspergillus oryzae ox-amylase, porcine pancreatic a-amylase, Triticum aestivum (wheat seed) a-amylase inhibitor, D-glucose, D-galactose, lactose, D-mannose, maltose, maltotriose, maltooligosaccharide, pullulan (limit dextrin)

Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii

“…Bacteria were cultured for 12 h, centrifuged, the cells were collected, washed and resuspended in PBS (0.01 M sodium phosphate bu¡er and 0.154 M NaCl (pH 7.3)). Puri¢ed amylase [2] (20 Wg) was added to the cells (at 1U10 10^2 U10 10 cells ml 31 ), and kept at room temperature for 1 h. The cells were then removed by centrifugation at 5000Ug for 10 min. The remaining amylase activity in supernatants was measured using an amylase activity kit according to the manufacturer's instructions (Sigma).…”

“…the resulting membranes were blocked with non-fat dry milk and incubated with lyophilized human parotid saliva (HPS) or puri¢ed amylase [2]. After washing, blots were incubated with rabbit anti-amylase, washed and incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase (Promega, Madison, WI, USA) and developed with the ProtoBlot Western Blot AP system (Promega).…”

“…The streptococcal cells and supernatants were collected by centrifugation at 5000Ug for 10 min. Proteins in the supernatants were precipitated either with acetone, according to standard methods [16], or by the addition of 50 Wg ml 31 of puri¢ed salivary amylase [2], followed by incubation at room temperature for 1 h. The precipitate containing amylase-binding streptococcal proteins complexed with amylase were collected by centrifugation at 5000Ug for 10 min, and resuspended in PBS.…”

Enzymic activity of salivary amylase when bound to the surface of oral streptococci

“…Certain species of oral streptococci are known te bind salivary a-amylase to their cell surface [ 1,2], This interaction appears to be largely species specific [3,4], virtually irreversible under physiological conditions [1, 2,5], and partly inhibited by amylase substrates [2,5]. While the molecular mechanisms of the interaction are not fully understood, the amylase receptor of one organism, Streptococc'~s gordonii strain challis, has been partially characterised [5].…”

“…It could, for example, provide organisms with a mechanism for attaching to the tooth surface. In vivo the tooth is coated with a layer of adsorbed salivary proteins (the acquired pellicle), including amylase, and so binding to amylase in pellicle could provide one mechanism for attaching to the tooth surface [1, 2,6]. However, such a mechanism seems unlikely given the high affinity salivary amylase in solution has for its streptococcal receptor [1,2].…”