Comments on Adherence of Streptococcus mutans MEAD M. McCABE Division of Oral Biology, School Miami, Florida 33152, USA

of Medicine, University of Miami,

We do not agree with the cell adherence model proposed by Dr. Slade nor with the concept that cell-to-surface adherence and dextran-induced intercellular adherence in Streptococcus mutans are separate and dissociable traits. Our data indicate that cellto-surface and intercellular adherence are induced by similar mechanisms involving the same glucan receptor sites on the cell surface and differing only in the physical nature of the glucan bond, the relative strengths of the cell-glucan interactions, and the requirements for divalent cations. Thus, cell-to-surface adherence occurs when numerous cell-surface glucan receptors bind to adherent branched insoluble glucans, forming a highly stable cell-glucan complex, whereas intercellular adherence is mediated by the binding of the same glucan receptors to soluble glucans such as linear dextran and requires the formation of a stabilizing intercellular calcium bridge.1 Dr. Hutton Slade's model for cell-to-surface adherence suggests that cell-bound glucosyltransferase (GTF) serves as both the means by which cells synthesize adherent glucans and as the only cell-surface binding site for these adherent glucans. This model is not compromised by Kuramitsu's observation that GTF-free heat-killed S mutans cells will bind to a preformed layer of insoluble glucan deposited on a glass surface, using cell-free GTF preparations,2 since the model suggests that the cells bind to residual cellfree GTF that is already bound to the glucan layer. The model also suggests that dextran (linear a-1,6-glucan)-induced intercellular adherence is mediated by a separate dextran-specific binding site on the cell surface. The observation of mutant strains that readily agglutinate in the presence of sucrose, dextran, or soluble glucan but are not able to form adherent microbial deposits in vitro supports this suggestion.3

The Slade model predicts that cell attachment to a preformed layer of adherent insoluble glucan will not occur if GTF is absent from both the cell surface and the glucan layer, and that low molecular weight dextrans, which readily inhibit dextran-induced intercellular adherence,4 will not affect the binding of cells to the preformed glucan layer. However, our results do not confirm these predictions. Washed glucosegrown cells of S mutans 6715-49 do not bind to preformed layers of adher-ent insoluble glucan in the presence of 5 mM soluble dextran (Dextran T-10, Pharmacia) nor do they agglutinate in the presence of this low molecular weight dextran when induced with a larger dextran (60 to 90,000 daltons, Nutritional Biochemicals) In addition, cells that have been heat-treated to destroy cellsurface GTF (20 minutes at 100 C) bind equally well to preformed layers of adherent glucan that have been treated to destroy residual GTF activity (0.5 M HCl at 37 C for 30 minutes, or heating at 100 C for 30 minutes) and to various control glucan layers. These results indicate that GTF does not mediate the binding of S mutans cells to adherent glucan, and strongly suggest that the same cell-surface glucan binding site that mediates intercellular adherence by binding soluble glucan and dextran also mediates cell-to-surface adherence by binding adherent insoluble glucan. Efforts to describe the events leading to cell-to-surface adherence of S mutans are dependent almost entirely on in vitro assay techniques designed to measure adherence of cells to glass surfaces or wires in the presence of sucrose and to assess the effects of various experimental alterations of the cells on this process. These assay techniques can easily give rise to artifacts, thus producing a nonspecific inhibition of the adherence process without affecting the components

C226 Downloaded from jdr.sagepub.com at CARLETON UNIV on March 15, 2015 For personal use only. No other uses without permission.

Vol 55 1976

COMMENTS ON ADHERENCE OF S MUTANS

of the cell surface responsible for adherence. The initial stages in the adherence of S mutans cells to a smooth surface require the synthesis of adherent insoluble glucans by cells that are in close proximity to that surface and the binding of the cells to the adherent glucans. Assay conditions that prevent the close approach of cells to the surface will probably prevent adherence. It may be more than coincidental that, as reported here by Dr. Slade, many antiserums that prevent in vitro cell-to-surface adherence also induce aggregation of S mutans cells. Aggregated cells probably are not capable of adhering to a smooth surface both because they are physically unable to approach near enough to the surface and because they rapidly settle from suspension. Thus, an indirect mechanism, antiserum-induced aggregation, could inhibit cell-to-surface adherence without actually affecting the cell components involved in adherence. Several mutants of S mutans are capable of intercellular adherence but do not form adherent microbial masses on wires immersed in cultures supplemented with 5% sucrose.3 Since all of these mutants synthesized copious amounts of soluble glucan in sucrose cultures, high levels of soluble glucan probably accumulated in the adherence assay cultures. Soluble glucan will serve as an acceptor molecule in the GTF reaction sequence, causing the synthesis of additional soluble glucan by enzymes that otherwise would synthesize only insoluble glucan.5 6 It also blocks the binding of cells to insoluble glucan layers. Thus, mutations affecting the complement of GTF enzymes elaborated by the cells, resulting in increased synthesis of soluble glucans, could have produced all of the effects observed, including failure to form adhesive microbial deposits, decreased levels of alkali-soluble cell-bound glucan, and decreased virulence of the mutants.3,7,8 This possibility does not detract from the significant observations reported by these authors but is pertinent to understanding the adherence mechanism. Our suggestion that a single, specific glucan-binding receptor functions in both cellto-surface and intercellular adherence requires that such a receptor be isolated. A likely protein has been isolated and is currently under study. During the course of our studies of S mutans GTF, we developed an

C227

affinity chromatography technique for rapid, high-yield purification of GTF (400-fold purification, 70% recovery) from culture supernatants and cell extracts. The technique involves adsorption of GTF onto a column containing insoluble glucans extracted from sucrose-grown S mutans KI-R followed by elution of the enzyme from the thoroughly washed column with solutions of clinical dextran. This technique has also been used to isolate a glucan-binding protein from cell extracts and culture supernatants of S mutans KI-R and 6715. This glucan-binding protein does not possess catalytic activity toward sucrose or dextran, nor is it a glycoprotein, although GTF appears to be. It is electrophoretically distinct from GTF on both standard disc electrophoretic gels of 7% polyacrylamide, and SDS-polyacrylamide gels, and it can be separated from GTF by hydrophobic chromatography. The absence of GTF or other sucrose-utilizing enzyme activity from this protein, the observation that its yield from various crude preparations is independent of GTF activity in the preparations, and its specific binding of insoluble glucan and soluble dextran suggest that this protein is the glucan receptor that mediates cell-to-surface and intercellular adherence in S mutans. References 1. MCCABE, M.M., and SMITH, E.E.: Unpublished data. 2. KURAMITSU, H.K.: Adherence of Streptococcus mutans to Dextran Synthesized in the Presence of Extracellular Dextransucrase, Infect Immun 9: 764-765, 1974. 3. FREEDMAN, M.L., and TANZER, J.M.: Dissociation of Plaque Formation from GlucanInduced Agglutination in Mutants of Streptococcus mutans, Infect Immun 10: 189-196,

1974. 4. GIBBONs, R.J., and FITZGERALD, R.J.: DextranInduced Agglutination of Streptococcus mutans, and Its Potential Role in the Formation of Microbial Dental Plaques, J Bacteriol 98: 341-346, 1969. 5. MCCABE, M.M., and SMITH, E.E.: Location and Solubilization of Cell-Bound Dextransucrase of Streptococcus mutans, Fed Proc 1243, 1972. 6. MCCABE, M.M., and SMITH, E.E.: Origin of the Cell-Associated Dextransucrase of Streptococcus mutans, Infect Immun 7: 829-838, 1973. 7. TANZER, J.M.; FREEDMAN, M.L.; FITZGERALD,

Downloaded from jdr.sagepub.com at CARLETON UNIV on March 15, 2015 For personal use only. No other uses without permission.

C228

MC CABE

R.J.; and LARSON, R.H.: Diminished Virulence of Glucan Synthesis-Defective Mutants of Streptococcus mutans, Infect Immun 10: 197203, 1974. 8. NALBANDIAN, J.; FREEDMAN, M.L.; TANZER,

J

Dent Res Special Issue C

J.M.; and LOVELACE, S.M.: Ultrastructure of mutants of Streptococcus mutans with Reference to Agglutination, Adhesion, and Extracellular Polysaccharide, Infect Immun 10: 1170-1179, 1974.

Downloaded from jdr.sagepub.com at CARLETON UNIV on March 15, 2015 For personal use only. No other uses without permission.

Comments on adherence of Streptococcus mutans.

Comments on Adherence of Streptococcus mutans MEAD M. McCABE Division of Oral Biology, School Miami, Florida 33152, USA of Medicine, University of Mi...
177KB Sizes 0 Downloads 0 Views