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Cell Wall-Associated Protein Antigens of Streptococcus Salivarius: Purification, Properties, and Function in Adherence ANTON H

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Cell Wall-Associated Protein Antigens of Streptococcus Salivarius: Purification, Properties, and Function in Adherence ANTON H INFECTION AND IMMUNITY, OCt. 1982, p. 233-242 Vol. 38, No. 1 0019-9567/82/100233-10$02.00/0 Copyright C 1982, American Society for Microbiology Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence ANTON H. WEERKAMPt* AND TON JACOBS Department ofMicrobiology, Faculty of Science, University of Nijmegen, Toernooiveld, NL-6525 ED Nijmegen, The Netherlands Received 17 February 1982/Accepted 7 June 1982 Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatogra- phy, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycopro- tein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 x 106 to 3 x 106 as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function. The primary event in the initial colonization of a lectin-like mechanism has been proposed on a host by most indigenous and pathogenic bacte- the basis of the inhibition of attachment by ria is the specific attachment of bacterial cells to certain sugars. In previous studies we have used certain surfaces in the host (8). However, in Streptococcus salivarius as a model organism to most cases the precise nature of the bacterial study the relationship between surface proper- surface components which mediate these inter- ties of bacteria and the ability of bacteria to actions is not known. Consequently, the mode adhere to and colonize suitable surfaces in a host of interaction between these components and (30-33). ligands on the host tissues is still poorly defined. S. salivarius, which is a successful inhabitant Surface appendages of the bacteria, which are of the human oral cavity, colonizes preferably often referred to as fibrils, fimbriae, or pili, have the tongue dorsum and buccal epithelium (8, 30). been implicated in the initial attachment of vari- This organism is also able to attach to tooth ous gram-negative and gram-positive bacteria to surfaces by means of saliva-derived components host tissues and other bacteria (7, 9, 20). In (30), but it is found only in low numbers in dental many cases proteins have been suggested as the plaque in vivo (8). S. salivarius readily forms adhesion receptors on the bacterial surface, and aggregates with several oral anaerobic, gram- negative bacteria (31). Such interaction may promote a with t Present address: Department of Oral Biology, State Uni- symbiotic relationship (e.g., versity of Groningen, Ant. Deusinglaan 1, 9713 AV Groning- veillonellae [32, 33]) or may serve as a mecha- en, The Netherlands. nism to foster the primary colonization and 233 234 WEERKAMP AND JACOBS INFECT. IMMUN. protection of oxygen-sensitive anaerobes (29, dissolved in a small amount of 50 mM Tris buffer (pH was removed nature of the cell-bound re- 7.5). Insoluble material by centrifuga- 31). Recently, the tion. ceptors involved in these adhesion reactions was The clear preparation obtained from 1.8 g (dry partially elucidated. The adhesion of streptococci weight) of cell walls was chromatographed on a to human oral surfaces requires protease-sensitive DEAE-Sephadex A-25 column (37 by 3.5 cm) in the components (31), and different proteinaceous cell same buffer and eluted at a flow rate of 6 cm/h. After wall constituents mediate coaggregation with Veil- passage of the nonadsorbed material, the column was lonella alcalescens (31, 33) and Fusobacterium further eluted with 800 ml of a linear 0 to 0.5 M NaCl and Bacteroides species (31). The specific nature gradient in Tris buffer. Fractions were monitored for of the adhesion characteristics was corroborated absorbance at 220 nm and for the presence of antigens are absent in by rocket immunoelectrophoresis. Antigen-containing by the finding that specific proteins peak fractions were pooled and concentrated by ultra- the cell walls of mutants which are defective in filtration, using a PM30 filter (Amicon B.V., Ooster- certain adhesion properties (33). hout, The Netherlands). The fractions were purified Cell wall proteins similar to those found in S. further by gel filtration on Sepharose CL-2B columns salivarius have been detected in Streptococcus (50 by 1.5 cm) and subsequently by chromatography mutans and have received attention as possible on immunoaffinity columns (6 by 1.5 cm) containing immunogens for caries prevention (15). Three specific antibodies directed against cell wall antigens such proteins were isolated by Russell and co- to remove contaminant antigens. The latter technique workers (23-26, 37) and were shown not to was used instead of binding to the complementary of the low of these columns or to cross-react weakly immu- antibody because capacity cross-react only and because elution under extreme or denaturing nologically with S. salivarius (23, 24). Another conditions (e.g., elution by chaotropic agents or at a study suggested that some of these proteins may low pH) could thus be avoided. be covalently linked to the peptidoglycan struc- Immunoaffinity columns were prepared by covalent- ture of the cell wall (19). ly linking the immunoglobulin fractions of the specific In this paper we describe the purification and antisera to cyanogen bromide-activated Sepharose properties of three cell wall-bound protein anti- beads (Pharmacia Fine Chemicals) according to the gens of S. salivarius HB, two of which appear to specifications of the manufacturer. The immunoglob- be related to specific adhesion properties of this ulins were isolated by using DEAE-Affigel Blue (Bio- Rad Laboratories), concentrated and washed by using bacterium. a membrane filter (type PM30; Amicon Corp.), and finally dissolved in coupling buffer. We used 25 mg of immunoglobulin per g (dry weight) of gel. MATERIALS AND METHODS Antigens that were excreted into the culture medium Bacteria. S. salivarius HB and mutant strains HB-7 during growth were partially purified by ammonium and HB-V5 have been described previously (31, 33). sulfate precipitation of neutralized culture superna- Unless otherwise stated, these strains were grown in tants (70% saturation). The precipitate was dissolved batch cultures in a Trypticase soy broth yeast extract in 50 mM Tris buffer (pH 7.5), and the solution was medium for 16 h at 37°C (33). subsequently brought to 65% (vol/vol) with ethanol. The oral streptococcal strains that were used for The precipitate that formed was collected by centrifu- reference were from our laboratory culture collection. gation and dissolved in about 1% of the original culture Purification of antigens. Triton X-100-treated cell volume of Tris buffer. walls were prepared from S. salivarius HB by the Preparation of antisera. The production of antisera procedure described previously (33). Cell walls were against cell walls of S. salivarius HB in rabbits has solubilized by incubation with mutanolysin (Ml en- been described previously (33). Specific antisera were zyme), an N-acetylmuramidase produced by Strepto- prepared by adsorbing anti-HB serum onto whole cells myces globisporus (preparation generously donated by of mutants HB-V5 and HB-7 (10 mg [dry weight] of K. Yokogawa [36]). Stock solutions of the enzyme (1 cells per ml of antiserum). The specific sera showed mg/ml) were prepared by dissolving the lyophilized single precipitation lines on Grabar-Williams immuno- powder in 40 mM sodium phosphate buffer (pH 6.2) electrophoresis gels with complete cell wall digests of containing 0.05% sodium azide and blending with a strain HB. Vortex mixer. The slightly turbid solution was clari- Streptococcal group K typing serum was obtained fied by centrifugation for 15 min at 40,000 x g. The from Wellcome Reagents Ltd., Beckenham, England. final preparation exhibited a slight proteolytic activity Immunological procedures. Standard immunoelec- (28). The cell walls were treated with the enzyme in a trophoretic and immunodiffusion techniques were per- wall-to-enzyme ratio of 200:1 (wt/wt) for 24 h at 37°C. formed with agarose gels (-Mr = 0.13; Bio-Rad) made After centrifugation for 20 min at 40,000 x g, the in Tris-Veronal buffer (ionic strength, 0.1 M; pH 8.6) insoluble residue was incubated for 24 h in one-half of containing 0.02% sodium azide and 1.2 mM calcium the original volume of enzyme solution. This proce- lactate. Electrophoresis was carried out at 10°C and 10 dure solubilized 70 to 80% of the cell wall dry weight.
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