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Growing cells of Streptococcus mutans Ingbritt incorporate radioactive mevalonate into unsaponifiable lipid. Of the radioactive lipid 40% was shown by chromatography and mass spectrometry to be C(50) and C(55) prenol.
Realization of the important role of Streptococcus mutans in the initiation of dental caries has resulted in extensive studies of all its biologic characteristics. The ultimate goal is to control or eliminate it from the oral cavity. One means of accomplishing this is to reduce or eliminate its carbohydrate source. This study reports those carbohydrates which Streptococcus mutans cannot metabolize.
A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs.
Invertase activity from Streptococcus mutans GS-5 has been partially purified and shown to possess beta-fructofuranosidase specificity. The enzyme has a broad pH optimum between pH 5.5 and 7.5 and exhibits maximal activity at 37 C. Fructose, but not the glucose analogue alpha-methyl-d-glucoside, acts as a competitive inhibitor of the enzyme. None of the common glycolytic intermediates or adenine nucleotides had any significant effect on enzyme activity. A molecular weight of approximately 47,000 was estimated for the enzyme. The enzyme does not appear to be catabolically repressed by glucose nor inducible by sucrose. Higher specific activities of the enzyme are observed in fructose or glucose-grown cells compared to sucrose-grown cells. These results are discussed in terms of the regulation of invertase activity in vivo.
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.
We examined the effects of human whole salivary supernatant and parotid fluid on glucose uptake by Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, Actinomyces viscosus, Staphylococcus aureus, and Escherichia coli. The following three effects of saliva were observed: (i) inhibition of glucose uptake (S. mutans, S. sanguis), (ii) promotion of a transient, rapid (0 to 30 s) burst of glucose uptake (S. mutans, S. sanguis), and (iii) enhancement of glucose uptake (S. mitis, A. viscosus, S. aureus, E. coli). We observed no differences between the effects of whole salivary supernatant and the effects of parotid fluid. Heat treatment (80 degrees C, 10 min) of saliva or the addition of dithiothreitol abolished inhibition of glucose uptake. Supplementation of saliva with H(2)O(2) potentiated inhibition of glucose uptake. S. mitis and A. viscosus, which were stimulated by saliva alone, were inhibited by H(2)O(2)-supplemented saliva; 50% inhibition of glucose uptake by S. mutans and S. mitis required ca. 10 muM H(2)O(2) in 50% (vol/vol) saliva. Loss of the inhibitory action of saliva occurred at about 5% (vol/vol) saliva. Supplementation of saliva dilutions with SCN(-) and H(2)O(2) extended the inhibitory activity to solutions containing ca. 0.2% (vol/vol) saliva. We suggest that the salivary lactoperoxidase-SCN(-)-H(2)O(2) system is responsible for the inhibitory activity of saliva reported here. Furthermore, we concluded that lactoperoxidase and SCN(-) are present in saliva specimens in concentrations that exceed minimal inhibitory levels by factors of ca. 500 and 10 to 20, respectively. The resistance of A. viscosus, S. aureus, and E. coli to the inhibitory potential of saliva alone was probably due to the production of catalase by these organisms. The resistance of S. mitis may have been due to special effects of saliva on H(2)O(2) accumulation by this organism compared with S. mutans and S. sanguis. The basis of saliva-dependent enhancement of glucose uptake and the basis of promotion of a transient, rapid burst of glucose uptake are unknown. The role of the salivary lactoperoxidase-SCN(-)-H(2)O(2) system in the oral microbial ecosystem is discussed.
Streptococcus mutans secretes a sucrose-independent branalphang enzyme that utilizes isomaltosaccharides as donors for branalphang formation on dextran. Although the branching enzyme is necessary for the formation of extracellular polysaccharide complexes, the source of the donor for the enzyme is unknown. In this study, we purified a novel glucosyltransferase from S. mutans and characterized its properties. The glucosyltransferase was primer independent 1,6-alpha-D-glucan synthase, which produced oligo-isomaltosaccharides. The enzyme was thought to be a source of donor for the branching enzyme in S. mutans.
Five Streptococcus mutans pairs (serotype c S. mutans 10449 and four clinical isolates of S. mutans: 123.1, LG1, OMFA, T10B) were used to find out if the xylitol-resistant (XR) natural mutants of the corresponding xylitol-sensitive (XS) S. mutans parental strains differ in their growth patterns in saliva. The isogenic X natural mutants of the parental S. mutans strains were selected after sequential cultivations in the presence of xylitol and glucose. The XR/XS strains pairs were grown in individual and pooled glucose-supplemented filter-sterilized salivas (one to five sequential cultivations). The two salivas used represented subjects with good or poor support of the growth of S. mutans in vivo. Protease and peptidase activities were determined from the saliva growth media and cell suspensions. Salivary protein profiles were analyzed using SDS-PAGE and native IEF before and after the cultivations. The growth properties of the XR/XS S. mutans pairs were similar in both individual and pooled salivas. Sequential cultivation of all strains did not show any differences in growth patterns. XS strains were inhibited by the presence of xylitol (2% w/v) in pooled saliva, as shown for other glucose-supplemented media. Protease and peptidase activities of the XR/XS S. mutans pairs were low and of similar magnitude. Also, the general hydrolytic properties of most XR/XS S. mutans pairs appeared similar as judged by the small growth-induced changes in salivary protein profiles. In conclusion, saliva, the source of nutrients for salivary microorganisms in vivo, favored neither the XR nor the XS strains of S. mutans.
OBJECTIVE: To study the relationship between genetic diversity within Streptococcus mutans (C), and dental caries. METHODS: One reference strain (GS5) was used to establish the suitability of primers and the initial AP-PCR conditions. Isolates of Streptococcus mutans (C) were obtained from 19 caries-free and 40 caries-active patients, and were originally isolated on mitis-salivarius-bacitracin agar. Totally 278 isolates were biochemically and serologically confirmed as Streptococcus mutans (C). Chromosomal DNA was extracted from those isolates by Chelex. One random primer, OPA-05 was used as a template in PCR reaction. All amplification reactions were conducted in a volume of 20 microliters containing 2 microliters of 10 x reaction buffer, 200 mumol/L each of dATP, dCTP, dGTP and dTTP and 2.5 units of Taq DNA polymerase. We varied the concentrations of MgCl2 (1.5-4.5 mmol/L), template DNA (2-40 ng), and primer (0.3-0.5 mumol/L) to determine the optical conditions for AP-PCR. The temperature profile in a thermocycler was 35 cycles as follows: 94 degrees C for 1 min, at 36 degrees C for 2 min, at 72 degrees C for 2 min. The initial denaturation was at 94 degrees C for 5 min and the final extension at 72 degrees C for 5 min. RESULTS: The MgCl2 concentration in 4 mmol/L and the primer concentration in 0.5 mumol/L produced clearly distinct and reproducible fingerprints. We found 105 different patterns among the 278 isolates. In high-caries persons more than one genotype isolates of Streptococcus mutans (C) were colonized. CONCLUSIONS: Isolates of Streptococcus mutans (C) exist apparent genetic diversity. The genotypes of isolates might relate to differences in caries susceptibility.
Streptococcus mutans possesses the abilities to adhere to pellicle-coated tooth surfaces and to form acids - two characteristics associated with the cariogenicity of this micro-organism. De novo synthesis of insoluble glucan by S. mutans glucosyltransferase from sucrose is essential in the adherence process. Therefore, agents which interfere with the adherence ability of S. mutans would be useful for controlling dental caries. In the present report, we have summarized our recent findings concerning virulence factors of S. mutans and means for prevention of S. mutans-induced dental caries.
The sgp gene of Streptococcus mutans was recently detected immediately downstream from the dgk gene within the same operon. In this study, the sgp gene was subcloned into the pMAL-c2 vector and SGP (S. mutans G protein) was overexpressed in Escherichia coli as a fusion protein with the maltose-binding protein at a level of 40% of total cellular protein. One-step amylose affinity chromatography purification of this fusion protein yielded a product of approximately 95% purity. SGP was purified from this fusion protein following cleavage with protease factor Xa and DEAE-Sephacel chromatography. In nucleotide binding assays, recombinant SGP showed specific binding for GTP and GDP, but not ATP, CTP, and UTP, and also catalyzed efficient hydrolysis of GTP to GDP. Kinetic studies revealed that the SGP Km value for GTP in this reaction was approximately 5.9 microM. Mg2+ also served as a cofactor of SGP in this reaction. In vivo subcellular localization by immunogold labelling demonstrated that SGP was associated with both membrane and cytoplasmic fractions. SGP not only had structural similarities with other G proteins but also proved to have high-level intrinsic GTPase activity. Therefore, SGP appears to be a new member of the G protein superfamily and may participate in transmembrane signaling in the responses of S. mutans cells to environmental stimuli.
The objective of this equivalency study was to see if a colony of Streptococcus mutans placed into cavities in primary molar teeth produced pulpitis similar to an established pulpitis induction method using carious dentine. In two juvenile baboons (Papio ursinus), occlusal cavities were cut in all 16 primary molar teeth, followed by making a small pulpal exposure after which the cavity was swabbed with 37 per cent phosphoric acid. In one half of the teeth, fresh soft human carious dentine was placed over the pulpal exposure; in the remaining teeth the exposure was covered with a colony of Streptococcus mutans in agar. All the cavities were restored with unlined light-cured composite resin. After 14 days specimens were harvested and examined under the light microscope with the examiner blind to the induction method. In both groups of teeth there was recognisable pulp, hyperaemia, micro-abscesses in the pulp and peri-apical abscesses. Reactions to soft caries were more severe than to Streptococcus mutans. The results show that Streptococcus mutans placed in a cavity with an exposure produces comparable pulpitis to fresh soft human carious dentine in the same type of cavity and that both methods produce pulpitis suitable for testing pulpotomy or pulpectomy treatments.
Monkeys were immunized with Streptococcus mutans by a number of routes in an attempt to elicit exclusively a secretory immunoglobulin A (IgA) response. Antibody responses were detected by a sensitive radioimmunoassay. Monkeys primed subcutaneously and boosted submucosally with formolized cells of S. mutans had high serum IgG, IgA, and IgM radioimmunoassay titers and only slight salivary IgG titers. Instillation of killed cells of S. mutans into the right parotid salivary duct elicited good IgG, IgA, and IgM responses in both the right parotid saliva and serum, but only a weak IgM response was detected in the left parotid saliva. Administration of killed cells of S. mutans in enterically coated capsules did not elicit a detectable antibody response or have a discernible effect on the antibody response to subsequent immunization by instillation. No increase in antibody titer was detected in the serum or whole saliva from monkeys orally immunized with enterically coated capsules containing viable S. mutans or in the serum, whole saliva, or intestinal contents from monkeys immunized with uncoated capsules containing killed cells of the same organism. These results do not support the concept that oral immunization with S. mutans is effective in stimulating a generalized secretory IgA response in primates.
A ribosomal preparation from Streptococcus mutans 6715 was characterized for its ability to induce an immune response in gnotobiotic rats which was protective against S. mutans-induced dental caries. Animals injected in the salivary gland region with the S. mutans ribosomal vaccine developed significantly higher (P less than 0.01) salivary IgA and serum IgG antibody activities against whole S. mutans cells and ribosomal preparations than nonimmunized rats. Vaccinated animals had significantly lower (67%; P less than 0.01) levels of S. mutans adherent to their molar surfaces than the control rats after infection with the homologous, cariogenic S. mutans. The immunized animals had significantly fewer (P less than 0.01) carious lesions on their buccal, sulcal, and proximal molar surfaces than the nonimmunized rats following challenge with the virulent organism. Animals injected with the ribosomal preparation developed salivary IgA and IgG antibodies with specificities to various cell surface-associated antigens such as lipoteichoic acid and glucosyltransferase, suggesting that the observed protection may be due to antibodies against cell surface contaminants of the ribosomal vaccine. These results are the first demonstration that a ribosomal preparation from S. mutans protected rats from caries formation after challenge with the homologous, virulent S. mutans.
Enolase from Streptococcus mutans has been purified to homogeneity by a three-step procedure. As shown by analytical ultracentrifugation and polyacrylamide gel electrophoresis under denaturing conditions, the purified enzyme is an octamer with molecular weight Mr = 360 kDa, composed of eight identical subunits with Mr = 45 kDa. The kinetic parameters of S. mutans enolase in the presence of 1 mM Mg2+ are Aspec = 130 IU x mg-1 and KM = 0.44 mM as determined by steady-state experiments in the D-glycerate 2-phosphate to enolpyruvate phosphate reaction. Enzymatic activity is inhibited noncompetitively by fluoride in the range between 0 and 10 mM NaF yielding Ki = Kii = 1.39 mM, but inhibition characteristics become competitive when tested above 10 mM. In the presence of only small amounts of phosphate (0.5 mM) the inhibitory effect of fluoride is enhanced dramatically yielding Ki = 0.26 mM. Inhibition by NaPO3F is competitive with Ki = 0.9 mM, indicating that free fluoride ions in combination with phosphate are more effective in inhibiting S. mutans enolase. It can be concluded that inhibition of enolase by fluoride in combination with phosphate can influence glycolysis in S. mutans and so reduce acid production or even growth rate, thereby leading to potential anticariogenic effects.
Previous studies carried out in our laboratory showed that Streptococcus mutans--a cariogenic oral bacteria--did not metabolize an anticariogenic sugar alcohol, xylitol, even after a 10-month adaptative period with monthly transfers to a fresh medium. Due to the potential risk to adaptation observed in bacterias, it was studied the behavior of Streptococcus mutans (Strain 1161, Ingbritt) exposed to the polyol during 7 months, without monthly transfers to a new medium. After 7 months the cells were monthly transferred to a fresh medium for 7 more months. The cells were maintained and grown in a Trypticase-soytone-base medium without dextrose which contained xylitol (0.25 g/100 mL) or no sugar added. The control was represented by cells of Streptococcus mutans growing in Trypticase- soytone-base medium containing dextrose (0.25 g/100 mL). The growth pattern in the presence of xylitol was similar to that obtained in the cultures without sugar added, but it was 63-78% lower when compared with the control. The final pH in the cultures with xylitol was around 6.0; in the control it was very low (4.69 +/- 0.12). When the cells maintained in xylitol were transferred to the medium containing dextrose, the growth pattern was similar to that of the control. Any cultures fermented xylitol; the pH of the fermentation medium remained around 6.00 when the xylitol was present. No uptake of 14C-xylitol was observed and the activity of the enzyme xylitol dehydrogenase could not be detected with the experimental procedure used. The present study confirmed the Streptococcus mutans inability to metabolize xylitol, even after a prolonged adaptative period in the sugar alcohol. Moreover, it demonstrated that xylitol could be considered an inert substance to Streptococcus mutans since the cells were viable in the presence of the xylitol, or in the medium without sugar added.
An inhibition factor from Streptococcus mutans strain C3603 (serotype c) was purified and isolated, and its properties indicated that it was a bacteriocin. Bacteriocin C3603 is a basic protein with a pI value of 10 and a molecular weight of 4,800. The activity of this bacteriocin was not affected by pH over a range of 1.0 to 12.0 or by storage at 100 degrees C for 10 min at pH 2.0 to 7.0 or storage at 121 degrees C for 15 min at pH 4.0. Pronase; papain, phospholipase C, trypsin, and alpha-amylase had no effect on the activity of the bacteriocin, whereas alpha-chymotrypsin and pancreatin were partially active against it. Bacteriocin activity was greater against certain S. mutans strains of serotypes b, c, e, and f than against certain S. mutans strains of serotypes a, d, and g. Bacteriocin C3603 was also effective against selected strains of S. sanguis, S. salivarius, S. bovis, S. faecium, S. lactis, Lactobacillus casei, L. plantarum, L. fermentum, Bifidobacterium bifidum, Bifidobacterium longum, Propionibacterium acnes, and Bacteroides melaninogenicus, but it was not effective against certain strains of Escherichia coli, Klebsiella pneumoniae, Corynebacterium parvum, and Candida albicans. The inhibition of S. mutans strains BHT and PS-14 by bacteriocin C3603 was found to be due to the bacteriocidal activity of the bacteriocin. When water or a diet containing bacteriocin C3603 was consumed by gnotobiotic and specific pathogen-free rats infected with S. mutans PS-14, the caries score was found to be significantly reduced.
Streptococcus mutans glucan-binding protein A (GbpA) has sequence similarity in its carboxyl-terminal domain with glucosyltransferases (GTFs), the enzymes responsible for catalyzing the synthesis of the glucans to which GbpA and GTFs can bind and which promote S. mutans attachment to and accumulation on the tooth surface. It was predicted that this C-terminal region, comprised of what have been termed YG repeats, represents the GbpA glucan-binding domain (GBD). In an effort to test this hypothesis and to quantitate the ligand-binding specificities of the GbpA GBD, several fusion proteins were generated and tested by affinity electrophoresis or by precipitation of protein-ligand complexes, allowing the determination of binding constants. It was determined that the 16 YG repeats in GbpA comprise its GBD and that GbpA has a greater affinity for dextran (a water-soluble form of glucan) than for mutan (a water-insoluble form of glucan). Placement of the GBD at the carboxyl terminus was necessary for maximum glucan binding, and deletion of as few as two YG repeats from either end of the GBD reduced the affinity for dextran by over 10-fold. Interestingly, the binding constant of GbpA for dextran was 34-fold higher than that calculated for the GBDs of two S. mutans GTFs, one of which catalyzes the synthesis of water-soluble glucan and the other of which catalyzes the synthesis of water-insoluble glucan.