Radioimmunoassay of class-specific antibodies to Streptococcus mutans in monkey serum and saliva.

A radioimmunoassay (RIA) has been developed to measure class-specific antibodies to Steptococcus mutans in the serum and saliva of monkeys (Macaca fascicularis). Anti-human immunoglobulin antibodies purified by affinity chromatography on immobilised monkey immunoglobulins and labelled with 125I were employed. Formolised cells of S. mutans and an extract of culture supernatant adsorbed to polystyrene wells were used as solid-phase antigens. The coefficients of variation for IgG, IgA and IgM assays were less than or equal to 10% for both antigen systems. Two monkeys were immunised with formolised cells of S. mutans by subcutaneous injection and subsequent instillation of bacterial cells into their right parotid ducts. IgG, IgA and IgM antibody responses to S. mutans in samples of serum and saliva were quantitated by RIA. Immobilisation of purified components of S. mutans on polystyrene wells enabled the measurement of antibody response to a number of antigens to be made. The RIA is a sensitive, reproducible and quantitative method of measuring serum and salivary antibody responses in monkeys.     

Naturally occurring secretory immunoglobulin A antibodies to Streptococcus mutans in human colostrum and saliva.

Human colostrum, parotid saliva, and serum were assayed for the presence of naturally occurring antibodies to five serotypes of Streptococcus mutans. Appreciable levels of agglutinins to strains AHT, BHT, 10449, 6715, and LM-7 (groups a leads to e, respectively) were detected in normal colostrum and saliva, whereas relatively low levels were found in serum. No agglutinins could be detected in the colostrum or saliva of immunodeficient patients. Molecular sieve chromatography of the colostrum on Sephadex G-200 revealed agglutinin activity in the secretory immunoglobulin A (s-IgA)-rich fraction only. Titration of purified colostral s-IgA confirmed the IgA nature of this agglutinating activity. Indirect immunofluorescence tests with anti-s-IgA, -IgG, and -IgM revealed S. mutans specificity only in the s-IgA class. The presence of s-IgA antibodies to indigenous oral microorganisms in colostrum, as well as in saliva, suggests that antigenic stimulation occurs at a site remote from the oral mucosa.     

Class-specific antibodies to Streptococcus mutans in human serum, saliva and breast milk

Previous techniques used for the detection and quantitation of antibodies in body fluids may be inappropriate where only small volumes are available, or may not be sensitive enough to detect low levels of specific antibodies. An indirect ELISA technique has successfully been employed to estimate class-specific antibody levels to Streptococcus mutans in serum and secretions in a group of mothers and their neonates, and an attempt has been made to relate such levels to the presence or absence of active caries in the mothers. A high maternal serum IgG antibody level appears to exert a protective effect against dental caries. Antibody levels in maternal saliva and colostrum/breast milk showed no differences between the 2 groups. The presence of active caries in mothers was associated with an elevated IgA antibody level in neonatal saliva. Although ELISA permitted the detection of low levels of antibody in the small volumes of neonatal saliva collected, a further increase in sensitivity and specificity of the assay would be advantageous.     

Characteristics of biofilm formation by Streptococcus mutans in the presence of saliva

Interactions between salivary agglutinin and the adhesin P1 of Streptococcus mutans contribute to bacterial aggregation and mediate sucrose-independent adherence to tooth surfaces. We have examined biofilm formation by S. mutans UA159, and derivative strains carrying mutations affecting the localization or expression of P1, in the presence of fluid-phase or adsorbed saliva or salivary agglutinin preparations. Whole saliva- and salivary agglutinin-induced aggregation of S. mutans was adversely affected by the loss of P1 and sortase (SrtA) but not by the loss of trigger factor (RopA). Fluid-phase salivary agglutinin and, to a lesser extent, immobilized agglutinin inhibited biofilm development by S. mutans in the absence of sucrose, and whole saliva was more effective at decreasing biofilm formation than salivary agglutinin. Inhibition of biofilm development by salivary agglutinin was differently influenced by particular mutations, with the P1-deficient strain displaying a greater inhibition of biofilm development than the SrtA- or RopA-deficient strains. As expected, biofilm-forming capacities of all strains in the presence of salivary preparations were markedly enhanced in the presence of sucrose, although biofilm formation by the mutants was less efficient than that by the parental strain. Aeration strongly inhibited biofilm development, and the presence of salivary components did not restore biofilm formation in aerated conditions. The results disclose a potent ability of salivary constituents to moderate biofilm formation by S. mutans through P1-dependent and P1-independent pathways.     

Comparison fo agglutinin titers for Streptococcus mutans in tears, saliva, and serum.

The agglutinin titers for three Streptococcus mutans serotypes (AHT, BHT, and 10449, representing serotypes a, b, and c, respectively) were measured in the saliva, tears, and serum of 19 human subjects. Naturally occurring S. mutans agglutinins were routinely present in all fluids tested in the absence of overt local stimulation by antigen. The immunoglobulin A nature of this secretory agglutinin activity was suggested by blocking with alpha heavy-chain-specific antiserum and by the demonstration of S. mutans-reactive immunoglobulin A in the saliva and tears by indirect immunofluorescence. This finding is consistent with stimulation and antigen commitment of immunoglobulin A precursor lymphocytes at remote sites and subsequent homing to the lacrimal system. The relationship of anti-AHT agglutinins to anti-10449 agglutinins differed among the body fluids tested. The tears had more agglutinins for strain AHT than for strain 10449, whereas the reverse was true for saliva and serum. A possible explanation is local antigen-driven expansion of AHT-reactive committed lymphocytes in the lacrimal tissues.     

Specific method for the purification of Streptococcus mutans dextransucrase.

A convenient and rapid method for the purification of Streptococcus mutans dextransucrase is described. Affinity chromatography, on a column containing insoluble dextran purified from a culture of S. mutans 6715-49, gave an almost 300-fold purification, with 76% recovery of enzyme. Subsequent hydrophobic chromatography on butyl-agarose increased the overall enzyme purification to more than 1,000-fold, with a 65% recovery of activity. Two components of the dextransucrase activity were separated during hydrophobic chromatography. Both synthesized insoluble glucan as their major product and were capable of synthesizing soluble glucan in the presence of exogenous soluble dextran. However, the major enzyme component, which coeluted with a catalytically inert, dextran-binding protein, was greatly stimulated by exogenous soluble dextran, whereas the second enzyme component was not.     

Effect of human saliva on the fluoride sensitivity of glucose uptake by Streptococcus mutans.

The fluoride (F) sensitivity of glucose uptake by whole cell suspensions of streptococcus mutans in the presence and absence of human whole salivary supernatant was studied. It was observed that dithiothreitol (DTT) and other thiols markedly reduced the F sensitivity of cells when saliva (50%, vol/vol) was present during glucose uptake. In the absence of saliva, cells were sensitive to 2 to 2.5 mM F regardless of the presence of thiols. Supplementation of cells in phosphate or tris(hydroxymethyl)aminomethane-hydrochloride buffers with physiological concentrations of calcium or phosphate had no effect on the F sensitivity of the organism. Experiments with permeabilized cells suggested that thiols themselves had no direct effect on the F sensitivity of enolase (a principal F target). Cells pretreated with DDT subsequently exhibited decreased F sensitivity when examined in the presence of saliva but not in the absence of saliva. Cells pretreated with whole salivary supernatant were found to be subsequently less sensitive to F in the absence of saliva during glucose uptake. Furthermore, in cases where cells were pretreated with saliva, subsequent additions of DDT were unnecessary to obtain maximal reduction in the F sensitivity of glucose uptake. It was concluded that the saliva-dependent reduction in F sensitivity of glucose uptake was not due to sequestration of available F by salivary constituents. The data suggest that a salivary component(s) interacts directly with the microorganism in some manner which results in reduced F sensitivity of the process under study. Possible mechanisms of saliva action are discussed.     

Activity of two Streptococcus mutans bacteriocins in the presence of saliva, levan, and dextran.

The extracellular dextrans produced from sucrose by Streptococcus mutans strains BHT and GS-5 did not prevent the synthesis or release of active bacteriocins by these two strains. In addition, several streptococci that were genetically sensitive to these bacteriocins, and that could synthesize a variety of extracellular dextrans and levans from sucrose, remained phenotypically sensitive when grown in the presence of sucrose. Bacteriocin activity was not altered by treatment with high-molecular-weight dextran or by human saliva. The bacteriocins produced by, and active against, S. mutans thus appear to be capable of acting in vivo and may play a role in regulating the bacterial ecology of the oral cavity.     

Adsorption of parotid saliva proteins and adhesion of Streptococcus mutans ATCC 21752 to dental fiber-reinforced composites

The use of fiber-reinforced composites (FRC) in dentistry has increased during recent years. In marginal areas of crowns and removable partial dentures the fibers may become exposed and come into contact with oral tissues, saliva, and microbes. To date, few articles have been published on oral microbial adhesion to FRCs. The aim of this study was to compare different FRCs, their components, and conventional restorative materials with respect to S. mutans ATCC 21752 adhesion and adsorption of specific S. mutans binding proteins. Surface roughness of the materials was also determined. Four different FRCs, a restorative composite, and a high-leucite ceramic material were studied. Polyethylene FRC was found to be significantly rougher than all other materials. Aramid FRC also showed higher surface roughness in comparison with all materials but polyethylene FRC. Without a saliva pellicle, adhesion of S. mutans coincided with surface roughness and polyethylene and aramid FRC promoted S. mutans adhesion better than the other smoother materials. In the presence of salivary pellicle, ceramic and polyethylene FRC bound more bacteria than the other materials studied. Higher quantities of S. mutans binding proteins in the pellicles may in part account for the higher S. mutans adhesion to saliva-coated ceramic and polyethylene FRC.     

Expression of Streptococcus mutans gtf genes in Streptococcus milleri.

The Streptococcus mutans glucosyltransferase (GTF) genes gtfB and gtfC were ligated into Escherichia coli-streptococcus shuttle plasmids and introduced into Streptococcus milleri. gtfB transformant KSB8 formed an S. mutans-like rough colony on mitis salivarius agar and expressed an extracellular GTF-I, of 158 kDa, and two cell-bound GTF-Is, of 158 and 135 kDa. gtfC transformant KSC43 formed a semirough colony on mitis salivarius agar and expressed primarily an extracellular GTF-SI, of 146 kDa, and two cell-bound GTF-SIs, of 146 and 152 kDa. The extracellular GTFs from KSB8 and KSC43 were purified and characterized. The two types of GTF also reacted specifically with monoclonal antibodies directed against each enzyme. Both enzymes synthesized significant amounts of oligosaccharides, consisting primarily of alpha-1,6-glucosidic linkages, as well as water-insoluble glucans, containing alpha-1,3-glucosidic linkages. Insoluble-glucan-synthesizing activities of both enzymes were stimulated (three- to sixfold) by the addition of dextran T10 and were inhibited in the presence of 1.5 M ammonium sulfate. The Km(s) for sucrose and the optimal pHs were also similar for both enzymes. However, when the transformants were grown in Todd-Hewitt broth supplemented with sucrose, KSC43 cells, expressing GTF-SI activity, adhered to glass surfaces in vitro, while KSB8 cells, expressing GTF-I activity, did not. These results are discussed relative to the potential role of the gtfB and gftC genes in S. mutans cariogenicity.     

Production of elevated levels of dextransucrase by a mutant of Streptococcus mutans.

A mutant (S19) of Sreptococcus mutans strain 6715 which produces elevated levels of dextransucrase (EC 2.4.1.5) was isolated. Soluble enzyme in culture supernatant solutions from S19 polymerized the glucosyl moiety of sucrose into alcohol-insoluble and water-insoluble glucans at a rate three to six times greater than that of the parent strain. Washed-cell suspensions of S19 also contained increased amounts of cell-associated enzyme. Adherence of S19 to glass in the presence of sucrose occurred at twice the rate of strain 6715. The Km values for sucrose and primer dextran were similar for the mutant and parent enzymes. Mutant S19 should facilitate studies on the mechanism of adherence of S. Mutans and the control of dextransucrase production by this bacterium.     

Plasmid-mediated transformation of Streptococcus mutans.

Streptococcus mutans GS-5 was transformed to erythromycin resistance with streptococcal plasmid pVA736. Transformation frequencies were higher with plasmids reisolated from transformed GS-5 cells relative to plasmid originally derived from S. sanguis Challis.     

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.     

Adherence of Streptococcus mutans and Streptococcus sanguis to salivary components bound to glass.

Adherence of radiolabeled Streptococcus mutans and Streptococcus sanguis to saliva-treated glass surfaces was studied under conditions which minimized bacteria-glass interactions. Treatment of glass with an alkylsilane solution decreased nonspecific bacterial adherence and enhanced adsorption of radiolabeled salivary components to these surfaces. Addition of Triton X-100 to the bacterial suspensions also reduced nonspecific adherence to siliconized glass, but did not affect adherence to salivary components attached to siliconized glass. Calcium stimulated S. mutans adherence to saliva-free glass, but inhibited adherence to saliva-treated glass. S. sanguis adherence to either saliva-free or saliva-treated glass was inhibited slightly at high calcium ion concentrations. Adherence of streptococci to saliva-treated glass exhibited saturation kinetics, and the numbers of binding sites on the experimental salivary pellicle and the affinity constants for bacteria-saliva attachment were determined. Preincubation of the streptococci with whole saliva decreased their capacity to adhere to saliva-treated glass, but not to saliva-free glass. Bacteria adherent to saliva-treated glass surfaces were readily desorbed by washing with saliva. The addition of homologous antisera, ammonium sulfate-precipitated immunoglobulins, or Fab fragments to the bacterial suspensions inhibited cell adherence to saliva-treated glass.     

Strains of Streptococcus mutans and Streptococcus sobrinus attach to different pellicle receptors.

We compared the levels of adsorption of Streptococcus mutans JBP and Streptococcus sobrinus 6715 to experimental pellicles formed from unsupplemented and glucosyltransferase (GTF)-supplemented saliva. Pellicles formed on hydroxyapatite beads from GTF or from saliva-GTF mixtures possessed detectable GTF activity. Low levels of GTF activity were also detected in clarified whole human saliva, but not in samples of submandibular saliva. The adsorptive behavior of S. mutans JBP to pellicles formed from saliva or saliva-GTF mixtures was strikingly different from that of S. sobrinus 6715. S. mutans JBP adsorbed in higher numbers to pellicles formed from whole or submandibular saliva than to buffer-treated hydroxyapatite under the assay conditions used, in which blocking with albumin was used. In contrast, S. sobrinus 6715 attached in lower numbers and did not show enhanced adsorption to pellicles prepared from saliva. Pellicles prepared from the high-molecular-weight mucin fraction of submandibular saliva effectively promoted adsorption of S. mutans JBP, but none of the saliva fractions tested enhanced the attachment of S. sobrinus 6715 above the levels of buffer controls. Exposure of pellicles which contained GTF to sucrose to permit in situ synthesis of glucan markedly enhanced attachment of S. sobrinus 6715 but not attachment of S. mutans JBP. Also, the presence of sucrose throughout the adsorption period did not enhance attachment of S. mutans JBP. Both organisms possessed cell-associated GTF, and GTF preparations derived from S. sobrinus 6715 and Streptococcus sanguis FC-1 behaved like GTF derived from S. mutans JBP. S. sobrinus 6715 attached in high numbers to dextran-treated hydroxyapatite, whereas S. mutans JBP did not. These observations suggest that S. mutans JBP cells possess an adhesin which binds to salivary components in the pellicles. In contrast, S. sobrinus 6715 cells appear to possess an adhesin which binds to glucan in the pellicles. Four additional strains of S. mutans and four additional strains of S. sobrinus behaved qualitatively like strains JBP and 6715, respectively, and thus the differences observed appear to be representative of these species. Collectively, our data indicate that S. mutans and S. sobrinus attach to different receptors in experimental pellicles.     

Identification and characterization of a nonimmunoglobulin factor in human saliva that inhibits Streptococcus mutans glucosyltransferase

Saliva contains an array of nonimmunoglobulin defense factors which are thought to contribute to the protection of the hard and soft tissue surfaces of the oral cavity by modulating microbial colonization and metabolism. Here we report the discovery of a putative innate defense factor in human saliva that inhibits the glucosyltransferase (GTF) of Streptococcus mutans, a virulence enzyme involved in oral colonization by this pathogen. The GTF-inhibiting factor (GIF) was initially identified as a nonimmunoglobulin salivary component that interfered with detection of antibodies to the glucan-binding region (GLU) of GTF by an enzyme-linked immunosorbent assay. This inhibitory activity was present in whole saliva and submandibular-sublingual saliva, but it was essentially absent from parotid saliva. GIF inhibited the recognition of S. mutans cell surface-associated GTF by specific antibodies but had no effect on antibodies to other cell surface antigens, suggesting that GIF specifically binds to GTF on S. mutans. GIF purified by size exclusion or affinity chromatography was used for biochemical and functional characterization. Analysis of GIF by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a high-molecular-weight glycoprotein after staining with Coomassie blue or Schiff's reagent. Heating and reduction with 2-mercaptoethanol of GIF resulted in the release of a approximately 58-kDa protein that was identified as alpha-amylase by Western blotting using anti-alpha-amylase antibodies. GLU bound blotted alpha-amylase, suggesting that the latter molecule is the GLU-binding component of the GIF complex. The ability of GTF to synthesize extracellular glucans was inhibited by GIF but not by uncomplexed alpha-amylase or an unrelated high-molecular-weight glycoprotein. In conclusion, our findings demonstrate that in human saliva, there is a high-molecular-weight glycoprotein-alpha-amylase complex which is capable of inhibiting GTF and may contribute to control of S. mutans colonization in the oral cavity.     

Pyruvate dehydrogenase activity in Streptococcus mutans.

Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.     

Adherence and Streptococcus mutans infections: in vitro study with saliva from noninfected and infected preschool children.

An in vitro adherence experiment was designed to mimic the transmission of Streptococcus mutans from mother to child to test the hypothesis that differences in initial adherence reflect differences in susceptibility to infection. The data show that the pretreatment of S. mutans cells with the saliva of the mother in a mother-child pair and the pretreatment of spheroidal hydroxyapatite with that of the child may result in combinations which counteract or foster the initial adherence to a varying extent. The findings indicate that such combinations may determine the risk of S. mutants infection.     

Linkage of sucrose-metabolizing genes in Streptococcus mutans.

Antibiotic resistance markers inserted adjacent to different cloned genes from Streptococcus mutans were used to determine the relative positions of these genes on the chromosome. The results showed that these genes, fru-1 and gbp, are closely linked to the gtfA-ftf-scrB cluster. However, gtfD was linked neither to this cluster nor to gtfB-gtfC.