Mutans Serotype Research Articles

Comparison of extracellular protein profiles of seven serotypes of mutans streptococci grown under controlled conditions.

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.

Monoclonal antibody to Streptococcus mutans type e cell wall polysaccharide antigen.

A monoclonal antibody against the polysaccharide antigen of Streptococcus mutans serotype e was prepared. It was found that beta-methyl-D-glucopyranoside and cellobiose markedly inhibited the precipitin reaction, whereas maltose showed no inhibition. The beta-glucosyl moiety of the type e polysaccharide seems to be the predominant antigenic determinant of the antigen.

Characterization of the serotype e polysaccharide antigen of Streptococcus mutans

The structure of the Streptococcus mutans serotype e polysaccharide was studied in order to determine the chemical basis of the immunological cross-reactions observed between it and the streptococcal group E polysaccharide. The chemical structure was established using methylation analysis, periodate oxidation, partial methanolysis and 13C NMR spectroscopy. The polysaccharide was found to consist of a polyrhamnose backbone of alternating 2- and 3-linked α- l-rhamnose units and sidechain β- d-glucosyl units linked to the 2-position of rhamnose units in the backbone. This structure of the oligosaccharide repeating unit of the S. mutans serotype e polysaccharide was identical to that of the group-specific polysaccharide of group E Streptococcus. Possible explanations for the previously reported differences in these two polysaccharides are discussed.

In vitro inhibition of adherence of Streptococcus mutans strains by nonadherent mutants of S. mutans 6715.

Four nonadherent mutants from Streptococcus mutans 6715 mutant UAB66 (serotype g) with similar phenotypes were shown to inhibit the adherence of adherence-proficient S. mutans serotypes c and g strains. One mutant, UAB108, was shown to inhibit adherence by wild-type strains representing serotypes a, d, and e as well. This inhibition of adherence was seen with pairs of strains grown in partially defined (PD) medium supplemented with 1% sucrose in both microtiter plates and glass tubes. The inhibiting factor was present in culture supernatant fluids of inhibiting strains grown in PD medium plus 1% sucrose and was heat stable. Ethanol precipitation of culture supernatant fluids of these strains yielded a water-soluble polymer which effectively inhibited the adherence of UAB66. This polymer, isolated from UAB108, was also shown to inhibit the adherence of UAB66 at lower concentrations than that needed to inhibit adherence with dextran T10. Partially purified glucosyltransferase, isolated from the culture supernatant fluids of glucose-grown UAB108, produced a water-soluble glucan which was shown to inhibit the adherence of UAB66 as well. The methods developed permit rapid screening for strains or mutants of strains or both that inhibit adherence or plaque formation or both by wild-type strains of S. mutans.

Structural studies of the rhamnose-glucose polysaccharide antigen from Streptococcus sobrinus B13 and 6715-T2.

The rhamnose-glucose polysaccharide antigens from the cell walls of Streptococcus sobrinus B13 and 6715-T2 (formerly Streptococcus mutans serotypes d and g, respectively) were structurally examined by using gas chromatography-mass spectroscopy. These data confirmed earlier chemical and serological studies suggesting that these polysaccharides had identical structures. The polysaccharides appeared to have a backbone of alternating 1,2- and 1,3-linked rhamnose units. Branching occurred at 1,2,3-linked rhamnose units. Side chains appeared to be composed of 1,2- and 1,6-linked glucose units with glucose as the only terminal carbohydrate.

Isolation and characterization of an extracellular glucosyltransferase synthesizing insoluble glucan from Streptococcus mutans serotype c.

A glucosyltransferase which synthesized insoluble glucan in polyacrylamide gel was isolated from the culture supernatant of Streptococcus mutans Ingbritt (serotype c) by ultrafiltration, ethanol fractionation, isoelectric focusing, and preparative gel electrophoresis. The enzyme preparation was electrophoretically homogeneous and immunologically distinct from the highly branched 1,6-alpha-D-glucan synthase and fructosyltransferase from the same strain and glucosyltransferases from serotypes a and g. The molecular weight was 99,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was 8.5. The enzyme had the optimum pH of 6.0 and Km value for sucrose of 9.4 mM. Besides the insoluble glucan with 96% 1,3-alpha linkage, this enzyme synthesized a considerable amount of diffusible glucan with 84% 1,6-alpha linkage, separately. This enzyme may be the one released from the enzyme aggregates by extracellular proteases, because the addition of extraneous trypsin to the crude enzyme preparation increased the amount of the enzyme species.

Antibody specificity and antigen characterization of rat monoclonal antibodies against Streptococcus mutans cell wall-associated protein antigens

Monoclonal antibodies to Streptococcus mutans OMZ175 (serotype f) cell wall-associated antigens (wall-extracted antigens [WEA]) were derived from the fusion of Lou C plasmocytoma rat cells (IR 983 F) and spleen cells from Wistar R inbred rats immunized with WEA. Four cell lines producing monoclonal antibodies directed against a component of S. mutans WEA have been established. All four monoclonal antibodies reacted only with two antigens of WEA from S. mutans OMZ175 by Western blotting and immunoprecipitation techniques, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot analysis of WEA showed that the four monoclonal antibodies recognized two related cell wall-associated proteins with apparent molecular weights of 125,000 and 76,000. Immunoprecipitation of whole cells with the monoclonal antibodies confirmed the surface localization of the two antigens. The ELISA and competitive ELISA were used to analyze the distribution of the epitopes on seven S. mutans serotypes. All S. mutans serotypes were found to express the recognized epitopes; however, different reactivity patterns could be distinguished among the various strains tested, and the four monoclonal antibodies reacted only weakly with S. mutans serotypes d and g.

Cloned gtfA gene of Streptococcus mutans LM7 alters glucan synthesis in Streptococcus sanguis.

Streptococcus mutans LM7 (Bratthall serotype e) chromosomal DNA was partially digested with EcoRI and ligated into the positive-selection plasmid vector pOP203(A2+). The ligation mixture was transformed into Escherichia coli, and transformants were selected for tetracycline resistance. Recombinant-bearing clones were screened for their ability to ferment raffinose, using the procedure of Robeson et al. (J. Bacteriol. 153:211-221, 1983). One raffinose-fermenting clone was isolated and found to contain a plasmid with an insert consisting of four EcoRI fragments totalling approximately 10.3 kilobases (kb). This strain was capable of growth on defined medium plus raffinose or sucrose and generated reducing sugars from a sucrose substrate. Southern hybridization analysis of the four EcoRI fragments revealed homology not only to S. mutans LM7 chromosomal DNA but also to S. mutans serotypes b, c, and f. Subcloning of this fragment array into a streptococcal E. coli shuttle vector indicated that a 2.4-kb EcoRI fragment was essential for sucrase activity. E. coli minicell experiments revealed a gene product of 55 kilodaltons. These data along with restriction endonuclease analysis and Southern hybridizations suggested that the cloned S. mutans LM7 gene was closely related to the gtfA gene cloned by Robeson et al. from S. mutans PS13 (Bratthall serotype c). The shuttle plasmid containing the 2.4-kb fragment was transformed into Streptococcus sanguis, which subsequently displayed increased sucrase activity in both intracellular and extracellular fractions. Elevated levels of synthesis of alcohol-insoluble and water-insoluble glucans were observed with crude extracellular fractions of the S. sanguis strain bearing the 2.4-kb fragment. An isolate cured of the shuttle plasmid plus the 2.4-kb fragment displayed wild-type S. sanguis glucan synthesis. In S. sanguis, this gtfA allele may play a role in glucan synthesis by interacting with extant high-molecular-weight glucosyltransferases.

Purification and characterization of a saliva-interacting cell-wall protein from Streptococcus mutans serotype f by using monoclonal-antibody immunoaffinity chromatography.

A rat monoclonal antibody, LO SM2, of the immunoglobulin M class, specific for a saliva receptor (SR) from Streptococcus mutans serotype f, was able to precipitate the SR from crude cell-wall-associated antigens (WEA) of this bacteria in presence of a detergent mixture. We have then used the technique of monoclonal-antibody immunoaffinity chromatography to purify the S. mutans SR. Pure SR was obtained from a crude WEA fraction with a single chromatographic step. The active SR could be eluted from the column in a highly purified form with 0.2 M-glycine/HC1, pH 2.8. The final yield was about 32% in terms of binding activity. Characterization of the SR by crossed immunoelectrophoresis, sodium dodecyl sulphate- or 4-30%-native-gradient-polyacrylamide-gel electrophoresis showed that the receptor is a single polypeptide chain of Mr approx. 74000. Native or denaturated forms of the SR adsorbed on to a solid support, such as nitrocellulose, are recognized by monoclonal antibody LO SM2, and both forms are still able to bind the ligand, saliva.

Immunochemistry of the Streptococcus mutans BHT cell membrane: detection of determinants cross-reactive with human heart tissue

Cell membranes of Streptococcus mutans BHT serotype b were prepared after glass bead disruption or mutanolysin digestion of whole cells. Immunoblot analyses of BHT membrane extracts revealed major polypeptides of 42,000, 46,000, 62,000, and 82,000 daltons, as well as several minor bands, to be reactive with rabbit anti-human heart immunoglobulins. Heart cross-reactive antigens have been reported in the cell walls and culture fluids of several S. mutans serotypes. This represents the first report of cell membrane-localized heart cross-reactive antigens in this oral pathogen. Positive enzyme-linked immunosorbent assay and immunoblot reactions were also obtained with heart tissue antigen and anti-BHT sera, indicating mutual cross-reactivity. The major cross-reactive component detected by immunoblotting of human heart extracts was a 69,000-dalton polypeptide.

Antigen analyses of serotypes of streptococcus mutans using a monoclonal antibody elaborated against serotype g polysaccharide antigen

A hybridoma (F4B) which produced a monoclonal antibody (mAb) specific for serotype g carbohydrate antigen (RR g) of Streptococcus mutans 6715 was obtained. The F4B mAb cross-reacted with purified carbohydrate antigens of serotype d (RR d) and serotype h (TCA h). In immunodiffusion tests, F4B mAb produced a stable precipitin band with RR g, while the band developed between the mAb and RR d/TCh h in the cold disappeared when incubated at room temperature. The immunoprecipitin reaction between F4B mAb and RRg was strongly inhibited upon addition of lactose.

Purification and properties of extracellular glucosyltransferase synthesizing 1,3-alpha-D-glucan from Streptococcus mutans serotype a.

Extracellular 1,3-alpha-D-glucan synthase (sucrose: 1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158 000 by SDS-PAGE and an isoelectric point of pH 5.2. The specific activity of the enzyme was 48.3 i.u. (mg protein)-1. The Km for sucrose was 1.2 mM and the activity was optimal at pH 6.0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-alpha-D-glucan by GC-MS.

Prevention ofStreptococcus mutans colonization by salivary IgA antibodies

The levels of salivary and serum IgA, IgG, and IgM antibodies to the seven serotypes (a-g) of Streptococcus mutans were established in 12 laboratory volunteers using a sensitive enzyme-linked immunosorbent assay. Salivary IgA antibody levels to the serotype c organism were significantly lower (P less than 0.005) than antibody levels to the other six serotypes of S. mutans. Similar results were found with a purified S. mutans serotype c carbohydrate. Serum IgG and IgM antibody titers to the serotype c whole cells were significantly higher (P less than 0.05) than to four other S. mutans serotypes (a, e-g). The abilities of S. mutans serotypes c and d to colonize molar tooth surfaces were examined in eight volunteers. S. mutans serotype d was cleared from the tooth surfaces within 24 hr of challenge, whereas S. mutans serotype c was detected in six of the eight volunteers after 2 weeks and in three of eight after 3 weeks. These results provide additional evidence for the role of salivary IgA antibodies in regulating S. mutans infection and suggest that the low levels of salivary IgA antibodies to S. mutans serotype c may contribute to the predominance of this serotype in the U.S. population.

A study on the aggregation of Streptococcus mutans. 2; The additive effect on the aggregation of Streptococcus mutans of salivary agglutinins and glucosyltransferase.

The effects of salivary agglutinins, calcium chloride, glucosyltransferase and sucrose in different combinations on the aggregation of S. mutans serotype c strain TH16 were examined using a new parameter of maximum slope for the quantitation of aggregating activity, and the interactions among them were statistically analyzed by the four-way analysis of variance. Salivary agglutinins, sucrose, calcium chloride and glucosyltransferase were concerned in the aggregation of S. mutans TH16 and the contribution of each factor was statistically significant. The interactions between these factors were additive except the synergetic interaction between sucrose and glucosyltransferase. Moreover, the contribution of glucosyltransferase and sucrose in the aggregation of S. mutans TH16 was 8 times as much as that of salivary agglutinins and calcium chloride.According to these results it is suggested that glucosyltransferase may play an important role as a dominant aggregating factor for S. mutans TH16 when sucrose exists, and its additive interaction with salivary agglutinins will become negligible.

Effect of fluoride on oral microorganisms. I. Effect of NaF on growth and dextransucrase activity of Streptococcus mutans.

The effects of NaF on the growth of Streptococcus mutans (St. mutans) and the activity of dextransucrase purified from St. mutans were examined in vitro. The minimum inhibitory concentrations (MIC) for a fluoride provided as NaF were lower than 180 ppmF without regard to St. mutans serotypes (a-g). NaF arrested bacterial growth at the concentrations of 180 and 360 ppmF bacteriostatically. No effects of fluoride on the synthesis of water-insoluble dextran were observed in the cell-freesystem.

Isolation and some properties of extracellular d-glucosyltransferases and d-fructosyltransferases from Streptococcus mutans serotypes c, e, and f

Extracellular d-glucosyltransferases (GTase) and d-fructosyltransferases (FTase) were isolated from Streptococcus mutans IB (serotype c), B14 ( e), and OMZ175 ( f) by chromatofocusing, followed by hydroxyapatite column chromatography. The GTases isolated from serotypes c, e, and f are basic proteins (pI 7.4). The serotype c and e enzymes have two protein components having M r 173 000 and 158 000 and the enzyme of the serotype f one component having M r 156 000. The GTases of all the serotypes showed a K m value for sucrose of 10–14m m and an optimum pH 5.5–6.0 for enzyme activity, and their activities were enhanced by the presence of primer Dextran T10. The α- d-glucans synthesized by the purified GTases are water soluble and primarily consist of (1→6)-α- d-glucosidic linkage (41–66 mol/100 mol) and α- d-(1→3,6)-branch linkage (6–20 mol/100 mol), but significant proportions of α- d-(1→3), α- d-(1→4), and α- d-(1→3,4) linkages (11, 6, and 14 mol/100 mol, respectively) were detected in the serotype c α- d-glucan. The isolated FTases of the serotypes c, e, and f are acidic enzymes (pI 4.6) and consist of two components having M r 84 000 and 76 000 for the serotype c enzyme, and 106 000 and 84 000 for the serotypes e and f enzymes, respectively. The K m value for sucrose was 6, 10, and 17m m for the serotypes c, e, and f enzymes, respectively, and the optimum pH of enzymic activity 5.5–6.0. Reactivity with Concanavalin A, susceptibility to acid hydrolysis, and paper chromatography of the hydrolyzates suggested that the water-soluble β- d-fructans synthesized by the purified FTases were of the inulin-type and had chemical structures somewhat different among the serotypes.

Characterization of monoclonal antibodies to Streptococcus mutans antigenic determinants I/II, I, II, and III and their serotype specificities.

Monoclonal antibodies (McAb) were developed to four protein components of Streptococcus mutans serotype c, some of which are significant in the protection against dental caries. The six McAb used in this investigation support the identities of streptococcal antigens (SA) I/II, I, II, and III. The specificities of these antigenic determinants were established both by direct binding and inhibition with the pure SA with a solid-phase radioassay. Whereas conventional antisera to S. mutans serotype c cross-react with serotypes c, e, and f (and g), McAb to serotype c-derived SA I/II react predominantly with serotype c and show some low-titer reactivity with serotype f. The slight cross-reactivity between S. mutants cells of serotypes c and f could be further differentiated by absorption of any of the three McAb to SA I/II with cells of serotype c. Parallel studies of McAb with cells of S. mutans and their ammonium sulfate-precipitated culture supernatants suggest that some SA determinants are retained predominantly on the cell surface, but others are readily shed into the culture medium, so that they are detected both on the cell surface and culture medium. Unlike polyclonal antibodies, McAb are capable of discriminating single antigenic determinants and can be applied to the study of shedding of antigens from microorganisms into the environment, such as the gut or gingival sulcus.

Glucose-sucrose-potassium tellurite-bacitracin agar, an alternative to mitis salivarius-bacitracin agar for enumeration of Streptococcus mutans.

An agar medium for selective recovery and enumeration of Streptococcus mutans was developed as an alternative to mitis salivarius-bacitracin (MSB) agar. Combinations of dyes, antibiotics, and tellurite were added to a nonselective medium which, because of its sucrose content, allowed easy recognition of S. mutans colonies. Candle jar incubation for 2 days, by comparison with anaerobic incubation, reduced background flora but did not diminish S. mutans recoveries from clinical samples. Quantitative comparisons were made of the simultaneous recoveries of a number of authentic S. mutans serotype representatives and fresh clinical isolates, using various glucose-sucrose-potassium tellurite-bacitracin (GSTB) formulations and mitis salivarius, MSB, and blood agars. Mitis salivarius counts were not detectably different from blood counts, but counts on MSB were distinctly lower. A formulation of the new medium containing 5% glucose 5% sucrose, 0.001% potassium tellurite, 0.3 U of bacitracin per ml (hence GSTB), and 2% agar gave recoveries nearly equal to those on mitis salivarius agar and much greater than those on MSB. The medium yielded readily recognized S. mutans colonies and facilitated detection of intracellular polysaccharide formers upon flooding with I2 reagent. Freshly isolated serotype c, E, and f colonies could often be distinguished from serotype d and g colonies, a distinction made reliable by testing for intracellular polysaccharide. A study of 300 salivary samples revealed GSTB to give significantly higher recoveries than MSB. About 72% of all samples were substantially underestimated for S. mutans with MSB, and 6.7% of samples were falsely negative for S. mutans with MSB. Recovery of background flora on GSTB was as low or lower than on MSB, and both types of agar could be stored for at least 9 weeks without notable change of selectivity. Thus, GSTB agar appears to be simple and reliable to use and requires no anaerobic incubation. Caution is voiced about interpretation of data previously reported which evaluated S. mutans on MSB agar.

Serological characterization of Streptococcus mutans serotype polysaccharide g and its different molecular weight forms.

The serotype polysaccharide g from Streptococcus mutans 6715 was found to cross-react with serotype polysaccharide a from S. mutans HS6 and serotype polysaccharide d from S. mutans B13. Double immunodiffusion experiments indicated that the serotype polysaccharide g consisted of the following: (i) the type-specific g site; (ii) a cross-reactive site g-a that was in common with polysaccharide a; (iii) a cross-reactive site g-d that was in common with polysaccharide d; and (iv) a cross-reactive site g-(a-d) that was in common with both polysaccharides a and d. Moreover, by a procedure involving several column chromatography steps, six polysaccharide-containing fractions showing reactivity with anti-g serum were found. By gel filtration, the molecular weight estimates of fractions LI, LII, LIII, SI, SII, and SIII were 2 X 10(6), 5 X 10(5), 6 X 10(4), 3 X 10(4), 1.4 X 10(4), and 1 X 10(4), respectively. Double immunodiffusion analysis indicated that LI, LII, and LIII contained the four antigenic sites of the putative polysaccharide g. LII also contained another additional immunodominant region, designated site x. The analysis also suggested that fraction SI lacked the type-specific site g, fraction SII lacked sites g and g-a, and fraction SIII lacked sites g, g-a, and g-d.

Clinical isolates of Streptococcus mutans serotype c with altered colony morphology due to fructan synthesis.

Streptococcus mutans MT6801 , MT6861 , and MT6879 , which form large mucoid colonies on mitis salivarius agar, were isolated from a mother and her two daughters. These isolates were identified as serotype c by immunodiffusion with serotype-specific antisera. The large colonies formed on sucrose-containing agar were found to contain water-soluble fructan . The cell-free fructosyltransferase ( FTase ) activity of the strains which formed large colonies was five to eight times higher than that of serotype c S. mutans which produced small, rough colonies typical of this serotype. Furthermore, greater quantities of fructan were synthesized from sucrose by growing cells of MT6801 when compared with MT8148 , a typical serotype c S. mutans. Glucosyltransferase and FTase could be isolated by chromatofocusing from culture supernatants of MT6801 and MT8148 . The FTase activity of both strains was eluted at pH 4.5, and glucosyltransferase was released by elution with an NaCl linear gradient. The eluted FTase activity of MT6801 was significantly higher than that of MT8148 . Strains MT6861 and MT6879 were also found to possess a similar property in terms of FTase activity. These results suggest that formation of large mucoid colonies by these strains is a consequence of high FTase activity.