Platelet aggregation by Streptococcus pyogenes.

Heat-killed group A Streptococcus pyogenes induced platelet aggregation in platelet-rich plasma. Aggregation was dependent upon the ratio of platelets to bacteria, with maximal aggregation occurring at 0.8 platelets per bacterium (final concentration, 300,000 per microliter). Inhibition of the reaction by 3 mM EDTA indicated it was a true aggregation and not merely adhesion and agglutination. Lactic acid dehydrogenase assays indicated lysis of platelets did not occur during a 6-min incubation period. Aggregation was inhibited in a dose-dependent manner by acetylsalicylic acid (100 microM to 10 mM) and quinacrine (15.6 to 250 microM), with no decrease in aggregation at the lowest concentration of inhibitor tested. S. pyogenes induced the release of [14C]serotonin, which was maximal (50%) at 2.4 min, when aggregation was nearly complete. Gel-filtered platelets were not aggregated unless fibrinogen (final concentration, 1.8 mg/ml) was included in the reaction mixture. Staphylococcus aureus, a group B streptococcus, and Escherichia coli were unable to induce aggregation in platelet-rich plasma under the conditions used for S. pyogenes.     

Insoluble glucan synthesis by Streptococcus mutans serotype c strains

Both dextransucrase and mutansynthetase activities have been purified from the culture fluids of Streptococcus mutans GS-5 (serotype c). Although homogeneous dextransucrase preparations normally synthesize little insoluble glucan, essentially all of the glucan synthesized by this enzyme in the presence of 1.5 M (NH4)2SO4 was water insoluble. Linkage analysis of the insoluble glucans indicated that the presence of NH4+ increased the portion of alpha-1,3-glucose linkages relative to alpha-1,6-glucose units in the product. Chromatofocusing of aggregated glucosyltransferase fractions synthesizing predominantly insoluble glucan yielded primarily dextransucrase activity separable from relatively low levels of mutansynthetase activity. The latter enzyme was detected only in 18-h assays and synthesized primer-dependent insoluble glucan, which was decreased in the presence of NH4+. In the absence of primer dextran T10, the addition of dextransucrase also stimulated insoluble glucan synthesis by mutansynthetase. Dextransucrase and mutansynthetase appear to be distinct enzymes, since the latter possesses a higher molecular weight (155,000 compared to 140,000), a much lower isoelectric point, and did not cross-react with antibody directed against dextransucrase. These results are discussed relative to the mechanism of insoluble glucan synthesis by S. mutans serotype c strains.     

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 alpha-L-rhamnose units and sidechain beta-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.     

Surface hydrophobicity, adherence, and aggregation of cell surface protein antigen mutants of Streptococcus mutans serotype c.

The pac gene of the serotype c strain Streptococcus mutans MT8148 encodes a cell surface protein antigen (PAc) of approximate 190 kilodaltons. The serotype c strain S. mutans GS-5 does not produce the 190-kilodalton PAc but produces a lower-molecular-weight protein that reacts with anti-PAc serum. The SphI-BamHI fragment of the pac gene was ligated with the S. mutans-Escherichia coli shuttle vector pSA3. The chimeric shuttle vector was transformed into strain GS-5, and two transformants (TK15 and TK18) were isolated. These transformants produced a large amount of cell-free and cell-bound PAc of 190 kilodaltons. No plasmid was isolated from these transformants, and the EcoRI fragments of their chromosomal DNA hybridized with the erythromycin resistance gene in the shuttle vector DNA, indicating insertion of the chimeric shuttle vector DNA into the chromosomal DNA. The cell hydrophobicity of strains TK15 and TK18 as well as PAc-defective mutants constructed by inserting an erythromycin resistance gene into the pac gene of strain MT8148 was analyzed. Strains MT8148, TK15, and TK18 were hydrophobic. On the other hand, strain GS-5 and PAc-defective MT8148 transformants were hydrophilic. Resting cells of the hydrophobic strains attached in larger numbers to saliva-coated hydroxyapatite than did the hydrophilic strains. Human whole saliva induced the aggregation of cells of the hydrophobic strains but not that of cells of the hydrophilic strains. These results suggest that cell surface PAc of S. mutans serotype c participates in attachment of the streptococcal cell to experimental pellicles.     

Impairment of melibiose utilization in Streptococcus mutans serotype c gtfA mutants.

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton sucrose-hydrolyzing enzyme. Analysis of S. mutans gtfA mutants revealed that the mutant strains were specifically impaired in the ability to use melibiose as a sole carbon source. S. mutans gtfA mutant strains synthesized less alpha-galactosidase activity inducible by raffinose than wild-type strains. Melibiose (an inducer in wild-type strains) failed to induce significant levels of alpha-galactosidase in the mutant strains. We hypothesize that melibiose use by S. mutans requires the interaction of the GtfA enzyme, or another gene product under the control of the gtfA promoter, with other gene product(s) involved in melibiose transport or hydrolysis.     

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.     

Antigens of Streptococcus mutans I. Characterization of a Serotype-Specific Determinant from Streptococcus mutans

A membrane-associated glycerol teichoic acid antigen has been isolated from Streptococcus mutans AHT and a similar antigen has been demonstrated to be present in each of the other Bratthall serotype a organisms studied. Trichloroacetic acid-extracted material was resolved into two phosphorus-containing antigenic fractions (B and C) by agarose chromatography. Fraction B was preliminarily identified as a phospholipid moiety with a glycerol-to-phosphorus ratio of 2:1, and fraction C showed a ratio of 1:1 indicative of a glycerol teichoic acid. This latter fraction also was associated with glucose, galactose, alanine, and fatty acids. Diglycerol triphosphate, the compound characteristically released from 1-3 phosphodiester-linked glycerol teichoic acids by alkaline hydrolysis, was isolated and characterized. Alanine was identified as its alkaline-labile, ester-linked D-isomer. A glyceride was isolated containing a disaccharide of glucose and galactose attached to the 2-hydroxyl group of glycerol. Hapten inhibition analysis demonstrated that beta-galactosides were the greatest inhibitors of the precipitin reaction (>75%), whereas glucose and its derivatives inhibited to a much lesser extent (<30%). Comparative immunodiffusion and immuno-electrophoresis analyses demonstrated that all six Bratthall serotype a organisms tested contained this antigenic determinant and that it was absent in serotypes b, c, and d. It is suggested that the common antigenic determinant of this serotype within S. mutans may be a beta-galactoside associated with a glycerol teichoic acid and possibly other polymers.     

Transfection of Streptococcus sanguis by phage deoxyribonucleic acid isolated from Streptococcus mutans.

Streptococcus sanguis ATCC 10556 cells were infected with free phage DNA of S, mutans strain PK 1. Two transformants were isolated which made colonies with large mucoid forms on mitis-salivarius agar. Both transformants had an increased ability to synthesize insoluble glucan and showed an adhesive nature on glass surfaces. These characteristics of the transformants bear a resemblance to S. mutans. These transformants had many physiological characteristics by which they could be recognized as S. sanguis. However, they resembled S. salivarius in forming a large amount of soluble fructan. Furthermore, the transformant cells did not produce ammonia from arginine, whereas their parent cells did.     

Platelet aggregation induced by antilymphocyte serum. 2. Two modes of the aggregation mechanism

The ability to inhibit the release reaction was found to correlate well with the ability to inhibit aggregation in washed human platelets exposed to antilymphocyte serum (ALS). However, the primary mechanism that contributes to ALS-induced aggregation in unwashed platelets appeared to be different from that found in washed platelets. Prostaglandin E1 and other release inhibitors were less effective in inhibiting aggregation in unwashed than in washed platelets. Salyrgan (mersalyl) in the presence of ALS or bovine fibrinogen produced an aggregation response in unwashed platelets that resembled the primary phase of aggregation. Two modes of the aggregation mechanism were evolved and are discussed.     

Specificity of antibodies induced by Streptococcus mutans during immunization against dental caries.

Protection against smooth surface dental caries was investigated in fifteen young rhesus monkeys which were immunized subcutaneously with Streptococcus mutans serotype c in Freund's incomplete adjuvant. Monkeys immunized with killed whole organisms developed significantly less caries than control animals. Monkeys immunized with pronase-treated cell walls developed significantly more caries than control animals while monkeys immunized with untreated cell walls showed no such enhancement of caries. Haemagglutinating and complement-fixing antibodies to cell walls and culture supernatant antigens (SN Ag) of S. mutans developed in the sera of all immunized animals to a similar degree. Antibodies to lipoteichoic acid and to an insoluble dextran preparation were found in all immunized animals and showed no relationship to the prevalence of caries. Antibodies to the serotype c polysaccharide were also found in animals immunized with whole cells and pronase-treated cell walls. However, precipitating antibody levels to partially purified antigens I/II and II, derived from SN Ag, but present also in cells, were related to the development of caries. Animals immunized with whole cells and with untreated cell walls developed a brisk antibody response to antigen I/II, while those immunized with pronase-treated cell walls responded more slowly. The results suggest that immunization may induce both caries reduction and enhancement, depending on the antibody response which is developed.     

Purification and immunological characterization of a rhamnose-glucose antigen from Streptococcus mutans 6517-T2 (serotype g).

The serotype antigens of Streptococcus mutans have been described as cell wall-associated polysaccharides. In this study, an additional wall polysaccharide antigen was purified and characterized from S. mutans strain 6715-T2, a mutant of 6715 (serotype g). Strain 6715-T2 lost the serotype antigen during animal passage. Rhamnose-containing carbohydrate fractions were solubilized from bacterial cells by extraction with 5% trichloroacetic acid at 4 degrees C for 18 h and with 0.01 N HCl at 100 degrees C for 20 min. Extracts were combined and purified on columns of diethylaminoethyl-Sephadex A-25 and Sephadex G-100. The purified sample contained 59% rhamnose, 31% glucose, 2.2% protein, and 0.24% phosphorus. The purified rhamnose-glucose polysaccharide (RGP/6715-T2) reacted strongly with antisera to whole cells of 6715-T2. Agar gel diffusion and comparative immunoelectrophoresis studies revealed that RGP/6715-T2 was serologically distinct from the serotype g and d polysaccharide antigens. These techniques also indicated immunological identity between RGP/6715-T2 and RGP/B13, a rhamnose-glucose polymer previously isolated from S. mutans B13, a serotype d strain. Antigen immunologically identical to RGP/6715-T2 was detected both in Rantz-Randall extracts from whole cells of S. mutans strains 6715, OMZ-65, and 6715-PT and in extracts from cells of 6715-T2 and C307, two mutant serotype g strains that lacked the serotype g antigen.     

Immunochemical Properties of Glucosyltransferases from Streptococcus mutans

Antiserum against purified mutansynthetase (EC 2.4.1.?) of Streptococcus mutans 6715 (serotype g), which is responsible for the synthesis of water-insoluble glucan (ISG) in the presence of both sucrose and water-soluble glucan, was prepared. The specificity of the antiserum was tested by using crude enzyme preparations (CEPs) of S. mutans strains of various serotypes. On immunodiffusion, the antiserum cross-reacted with CEPs from strains of serotypes a (HS-6 and AHT), d (OMZ176), and g (OMZ65 and KIR), but not with those from strains of serotypes b (BHT and FA-1) and c (GS-5 and Ingbritt). The antiserum inhibited the synthesis of ISG by crude or purified mutansynthetase of S. mutans 6715. The activities of ISG synthesis by CEPs from the strains antigenically related in the foregoing immunodiffusion were inhibited by the antiserum against strain 6715 mutansynthetase. The antiserum, however, also inhibited the enzyme activity of the strains of serotype b. The finding that the antiserum against purified dextransucrase of S. mutans HS-6 inhibited ISG synthesis by a CEP of strain HS-6 and also by CEPs of antigenically related strains suggested that dextransucrase activity is involved in ISG synthesis.     

Isolation and characterization of Streptococcus mutans mutants defective in adherence and aggregation.

A method was developed which enriched for mutants of Streptococcus mutans that exhibit defects in adherence to glass, aggregation, or both. Mutants were isolated from derivatives of strains PS14 (serotype c) and 6715 (serotype g) after mutagenesis with either ethyl methane sulfonate or nitrous acid. Cell survival after mutagenesis was kept above 1 to 2% to enhance the probability that mutants resulted from single mutational events. A total of 117 mutants were isolated; they also displayed non-wild-type colony morphology on mitis salivarius agar. These mutants were examined for (i) adherence and aggregation after overnight growth in sucrose-containing medium, (ii) aggregation of nongrowing cells in the presence of 200 microgram of sucrose per ml or 20 microgram of dextran per ml, and (iii) dextranase production on blue dextran agar plates. Although we isolated mutants which exhibited a variation from the parent strain in only one of the traits tested, the majority of mutants exhibited defects in two or more characteristics. Thirty-eight stable mutants of independent origin were categorized into 13 separate phenotypic groups.     

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.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Cariogenicity of a lactate dehydrogenase-deficient mutant of Streptococcus mutans serotype c in gnotobiotic rats.

A lactate dehydrogenase-deficient (Ldh-) mutant of a human isolate of Streptococcus mutans serotype c was tested in a gnotobiotic rat caries model. Compared with the wild-type Ldh-positive (Ldh+) strains, it was significantly (alpha less than or equal to 0.005) less cariogenic in experiments with two different sublines of Sprague-Dawley rats. The Ldh- mutant strain 044 colonized the oral cavity of the test animals to the same extent as its parent strain 041, although its initial implantation was slightly but not significantly (P greater than or equal to 0.2) less. Multiple oral or fecal samples plated on 2,3,5-triphenyltetrazolium indicator medium revealed no evidence of back mutation from Ldh- to Ldh+ in vivo. Both Ldh+ strain 041 and Ldh- strain 044 demonstrated bacteriocinlike activity in vitro against a number of human strains of mutans streptococci representing serotype a (S. cricetus) and serotypes c and e (S. mutans). Serotypes b (S. rattus) and f (S. mutans) and strains of S. mitior, S. sanguis, and S. salivarius were not inhibited. Thus, Ldh mutant strain 044 possesses a number of desirable traits that suggest it should be investigated further as a possible effector strain for replacement therapy of dental caries. These traits include its stability and low cariogenicity in the sensitive gnotobiotic rat caries model, its bacteriocinlike activity against certain other cariogenic S. mutans (but not against more inocuous indigenous oral streptococci), and the fact that it is a member of the most prevalent human serotype of cariogenic streptococci.     

Fluoride uptake by Streptococcus mutans 6715.

The short-term kinetics of fluoride uptake by cells from 20- to 22-h cultures of Streptococcus mutans strain 6715 were studied using rapid filtration and centrifugation techniques. Saline-suspended organisms were diluted with fluoride-containing solutions buffered at four different pH values (2.0, 4.0, 5.5, and 8.2). Fluoride disappearance from the medium was inversely related to pH and to the duration of the exposure at any given pH. The uptake was rapid and extensive at the lower pH values and decreased as the pH increased. Media fluoride concentrations subsequently increased; i.e., fluoride was released from the cells. The presence of glucose, cyanide, or iodoacetate did not influence the results. However, preincubation of the cells in fluoride-free buffers, followed by the addition of fluoride, reduced fluoride uptake markedly. Cell-to-media pH gradients were determined by the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione. Fluoride uptake was found to be a function of the magnitude of the pH gradient (P less than 0.001). It is hypothesized that fluoride uptake occurs by the diffusion of hydrogen fluoride and the subsequent trapping of ionic fluoride.     

Virulence of Streptococcus mutans: characterization of a serotype g antigen-defective mutants and its revertants.

A mutant of Streptococcus mutans 6715 HSR, which is defective in serotype antigen and designated C307, was shown to exhibit full virulence on buccal, sulcal, and proximal surfaces similar to that of 6715 HSR. In addition, this bacterium caused significant decay on the lingual surfaces. Its colonial morphology and certain biochemical activities which may be related to caries production were distinct from those of 6715 HSR. This mutant adsorbed to saliva-treated hydroxyapatite beads in greater amounts and aggregated in the presence of either sucrose or dextran in excess of that seen with the parent strain. The abilities of C307 to grow and to produce acid from sucrose and to adhere to glass surfaces were similar to that of 6715 HSR. Although revertants of C307 exhibited biological activities and a content of serotype g antigen similar to that of 6715 HSR, the virulence pattern was still unlike the parent strain. These results suggest that the serotype g antigen is not required for the adherence of cells to smooth surfaces or for caries formation and that the loss of this antigen may alter the surface of cells causing enhanced ability of the cells to aggregate and to adsorb to saliva-treated hydroxyapatite beads.     

A human salivary protein which promotes adhesion of Streptococcus mutans serotype c strains to hydroxyapatite.

The aim of this study was to investigate the nature of one of the factors in human submandibular-sublingual (SMSL) saliva which promotes the adhesion of Streptococcus mutans serotype c strains to hydroxyapatite (HA) surfaces. Gel filtration chromatography of SMSL saliva on Trisacryl GF2000 gave a void volume peak which contained the major fraction of adhesion-promoting activity for S. mutans JBP to HA. Maximum adhesion-promoting activity, however, eluted slightly later than the maximum 220-nm absorbance of the void volume peak. Gel filtration of the void volume material after treatment with sodium dodecyl sulfate (SDS) gave an early-eluting larger peak followed by a smaller peak with which the adhesion-promoting activity was associated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of relatively slowly migrating material associated with the larger inactive peak, presumably mucin, and a faster-migrating band(s) associated with the smaller active peak. SDS-PAGE indicated molecular weights in the range of 300,000 to 350,000 by extrapolation from size standards. Comparison of SMSL from five individuals showed the presence of single bands or double bands associated with adhesion-promoting activity, indicating genetic polymorphism. The active material did not resemble either secretory immunoglobulin A, based on SDS-PAGE and immunoassay, or fibronectin, based on SDS-PAGE, and also differed in molecular weight from salivary mucins and salivary constituents previously reported to promote aggregation of certain oral bacteria, but a relationship to these materials cannot be excluded. This adhesion-promoting material may play a significant role in the initial colonization of tooth surfaces by S. mutans strains.