Streptococcus sanguis surface antigens and their interactions with saliva

Abstract

Saliva-binding molecules of Streptococcus sanguis and their receptors were investigated. Streptococcal cell surfaces were extracted with a barbital buffer and examined immunochemically. Strains G9B and Blackburn, which adhere specifically to saliva-coated hydroxyapatite via immunologically related adhesins, possess 80-, 62-, and 52-kilodalton (kDa), and 52-, 42-, and 29-kDa polypeptides, respectively, which correlate with adhesion to saliva-coated hydroxyapatite. Nonadherent strains Adhand M-5 lack these antigens. In an immunoblot overlay, the putative adhesins bound to a 73-kDa receptor present in submandibular saliva but not in parotid saliva. G9B also contains a 160-kDa surface protein which bound to an unidentified receptor in both submandibular and parotid saliva samples. Blackburn barbital-extracted components bound to 78and 70-kDa receptors in parotid saliva. These bacterial-salivary interactions may be important in the regulation of oral ecology. * Corresponding author. MATERIALS AND METHODSBacteria and culture conditions. S. sanguis G9B and M-5 are reference strains originally isolated from dental plaque. Strain Blackburn (NCTC 10231) was obtained from R. M. Cole, National Institutes of Health, Bethesda, Md. Strain Adh-is a poorly adhering (see below) spontaneous mutant of G9B isolated from a chemostat culture (manuscript in preparation). After initial isolation, the strains were maintained at -70°C and subcultured only infrequently. The strains were characterized physiologically by the criteria of Carlsson (7) and Facklam (16) and fit the Facklam biotype I category. On the basis of the data of Rosan (32), strains G9B and Adhare serotype 1, whereas M-5 and Blackburn are serotype 2. All of these strains have approximately the same saliva-mediated aggregating activity (35).Cells were grown overnight at 37°C in Trypticase peptone broth (BBL Microbiology Systems, Div. Becton Dickinson and Co., Cockeysville, Md.) supplemented with 5 g of yeast extract (BBL) and 0.5% glucose as a carbon source.Saliva. Stimulated parotid saliva and submandibular saliva samples were used. Parotid saliva was collected in Curby cups from 15 individuals and pooled. Submandibular saliva was collected from one individual, previously found to have high adherence-promoting activity, by a plastic mouthpiece collector custom fit for the subject (5). The salivas were clarified by centrifugation (44,000 x g, 15 min) and either aliquoted and stored at -20°C or freeze-dried and stored at +40C.Adhesion assay. Bacterial adhesion to saliva-coated hydroxyapatite (SHA), an in vitro model of the tooth surface, and inhibition of adhesion by antibody were measured by the methods of Rosan et al. (34).Barbital extraction of bacteria. Cells were harvested and washed three times in H20 by centrifugation at 10,000 x g for 10 min at 40C. The pellet was suspended at 5 mg/ml in cold barbital buffer (2 mM sodium barbital, pH 8.6) and vortexed for 30 min at 40C. The cells were harvested by centrifugation (10,000 x g, 10 min, 4°C), and the supernatant was coll...

Streptococcus gordonii utilizes several distinct gene functions to recruit Porphyromonas gingivalis into a mixed community

“…Negative controls included blocking buffer alone to control for any non-specific binding of P. gingivalis to the nitrocellulose; and S. gordonii OB219 that lacks the Ssp adhesins and does not support P. gingivalis biofilm development (Demuth et al, 1996;Lamont et al, 2002). In addition, for each mutant showing a biofilm defective phenotype, a membrane with deposited S. gordonii cells was probed with anti-S. gordonii whole cell polyclonal antibodies (1:3000) (Lamont et al, 1988) and biotinylated secondary antibody to ensure equivalent numbers of S. gordonii were present on the nitrocellulose. To corroborate the fidelity of biotin labelled cells as a measure of P. gingivalis numbers, P. gingivalis cells were labelled with [ 3 H]-thymidine and numbers determined by scintillation counting of excised spots of the nitrocellulose (Lamont and Rosan, 1990).…”

Involvement of Porphyromonas gingivalis fimbriae in adherence to Streptococcus gordonii

“…The colutnn was washed with Tris buffer and eluted with a linear gradient of 0 to 0.3 M NaCl. Fractions were monitored spectrophototnetrically at 215 ntn and by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGF) (13,16) with the Bio-Rad silver stain (Bio-Rad Laboratories, Richtnond, CA). Satnples containing fimbriae were pooled, dialyzed overnight against 4 liters of 0.03 M Tris-HCl pH 8.0 and concentrated in an Atnicon model 52 filtration unit (Atnicon, Beverly, MA).…”

Isolation and characterization of a non‐adherent mutant of Streptococcus sanguis G9B

“…In contrast, neither the adhesion of the mutant nor the amount of A antigen detected in Adh was affected by growth rate. The A antigen contained polypeptides of 80, 62, and 52 kDa which we previously found to be associated with the adhesion of G9B to SHA (24). The evidence suggests the A antigen is the native form of the antigen complex responsible for the adhesion of S. sanguis G9B to salivary pellicle.…”

“…All of the studies reported here used the same pools of antisera and the IgG derived from these pools. Mouse anti-80-kDa serum was prepared using essentially the same protocol described previously for rabbit anti-80-kDa serum (24,25), In brief barbital extracts of G9B were subjected to SDS-PAGE (see below) and the 80-kDa band was eut out of the gel, macerated in 0.15 NaCI and mixed with an equal volume (ca. 0.2 ml) of Freund's complete adjuvant.…”