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Three glucan-binding proteins have been isolated from the extracellular fluid cultures of Streptococcus mutans serotype c. These proteins were adsorbed to glucans containing 1,3-alpha or 1,6-alpha bonds and linked to various chromatographic supports: they were eluted from columns by a dextran solution. Glucosyltransferase activity was associated with two of the glucan-binding proteins.
In Streptococcus mutans, enzyme II(scr) and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose. The scr regulon of S. mutans is composed of three genes, scrA and scrB, which code for enzyme II(scr) and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator. It was previously shown that expression of both scrA and scrB is similarly induced by sucrose. Mutation in the scrR gene resulted in increased expression of scrB relative to that in the wild-type strain. In this study, we employed DNA mobility shift and DNase I protection assays with a purified ScrR-histidine tag fusion protein to examine the DNA binding properties of ScrR to the promoter regions of the scrA and scrB genes. The results showed that ScrR bound specifically to the promoter regions of both scrA and scrB. Two regions with high affinity for ScrR in the promoter sequences of the scrA and scrB genes were identified by DNase I protection assays. One, O(C), which includes a 20-bp imperfect inverted-repeat sequence, is located between the two promoters, and the other, O(B), is located within the scrB promoter region containing a 37-bp imperfect direct-repeat sequence. Mutations of O(B) and O(C) resulted in constitutive transcription and expression of both the scrA and scrB genes. Our results indicated that S. mutans coordinates the activities of enzyme II(scr) and sucrose-6-phosphate hydrolase by transcriptional repressor ScrR binding to the promoter regions of the scr regulon.
To develop a bacterial demineralization model, we grew Streptococcus mutans biofilms in a constant depth film fermentor (CDFF) and studied the effects of sucrose pulsing frequency (SPF) in time on dentin demineralization. S. mutans biofilms were grown in dentin specimens with grooves and on dentin surface specimens for 20 days. During the experiments, 2% sucrose was pulsed either 4 or 8 times per day for periods of 30 min. Diluted brain-heart infusion medium containing 25 mM PIPES buffer and 1.5 mM CaCl2 was pulsed as the alternative growth medium. Specimens with intact biofilms were taken out on days 5, 12 and 20. The model was assessed by viable counts of the biofilm, mineral loss and lesion depth in the dentin specimens (by transversal microradiography) and pH measurements in the groove (by pH microelectrode). The results showed that biofilms formed on the dentin surface specimens were constant in viable counts for the low SPF, while this parameter tended to increase with time under the high SPF. Lesions with intact surfaces were formed and the lesion size increased significantly over time and increased significantly with increasing SPF. Typical Stephan curves were found after sucrose pulsing. The pH inside the groove returned to neutral under low SPF, but remained below 6.5 under high SPF. With the CDFF S. mutans biofilm model, lesions can be created in dentin within reasonable experimental time periods, as a result of the presence of a biofilm and in response to carbohydrate challenges.
The fluoride resistance and smooth surface adherence characteristics of Streptococcus mutans were examined using tooth model and radioactive cell assays. Resistance to 600 ppmF by S. mutans isolated from the plaque of radiation-induced xerostomia patients receiving daily topical applications of a caries preventive 1% NaF gel was transient. Resistance induced in vitro in two strains of S. mutans by exposure to gradually increasing levels of NaF was apparently permanent. Smooth surface adherence by both fluoride-sensitive and -resistant strains of S. mutans 6715 in a tooth model system was slightly diminished by 1% NaF gel. Fluoride-resistant strains retained 89 to 93% of their adherence capability in 600 ppmF, as determined by the cell radiolabeling assay.
The sucrose phosphotransferase system of Streptococcus mutans catalyzes the phosphorylation of sucrose to sucrose-6-phosphate with concomitant translocation of this disaccharide across the cytoplasmic membrane in reactions requiring intracellular phosphoenolpyruvate. Soluble proteins released by vigorous homogenization of cells with glass beads are shown to be necessary for the phosphoenolpyruvate-dependent phosphorylation of sucrose in combination with one or more proteins that remain tightly associated with the membrane fraction. We have partially purified phosphotransferase enzyme I and have purified a heat-stable phosphocarrier protein (HPr) to apparent homogeneity, by gel filtration and ion-exchange chromatography from the soluble fraction. HPr from S. mutans has an apparent molecular weight larger than that of Escherichia coli HPr but has properties similar to those of Staphylococcus aureus HPr. Furthermore, it appears to be partially complexed with a heat-stable enzyme III-like protein in cell-free fractions from S. mutans, and we also report the purification of this complex. Enzyme I from S. mutans is a protein (native Mr greater than 100,000) that cross-complements enzyme I from S. aureus. Preliminary characterizations of homogeneous HPr and its complex with the putative enzyme III are also presented.
Using morphological and biochemical criteria, it was possible for Streptococcus mutans to be isolated, in one case, from the bacterial plaques obtained from three test subjects. The colonies of this clearly identified species were characterized by a granular surface resembling frosted glass, had attached to them a shining bubble, and were seen to split mannite and sorbite in the absence of conversion of glycerol.
The prevalence of Streptococcus mutans and lactobacilli was determined in 62 18-month-old Dutch children with a cleft lip and/or palate. Plaque and saliva samples were collected, a dental examination was performed, and the parents were interviewed with a structured questionnaire regarding general health, dietary habits, fluoride exposure, and socioeconomic class. Appropriate dilutions of the plaque and saliva samples were cultured on selective media to count all viable bacteria, S. mutans and lactobacilli. S. mutans was detected in the saliva of 45% of the children, and lactobacilli was detected in 16%. Also, S. mutans was detected in 48% of the plaque samples and lactobacilli in 8%. Of all of the variables examined, consumption of more than three snacks and beverages between main meals was significantly associated with presence of S. mutans in saliva. Preoperative infant orthopedic treatment (i.e., wearing an acrylic plate from shortly after birth) was significantly associated with presence of lactobacilli in saliva. The presence of S. mutans in the plaque samples was also significantly associated with presence of lactobacilli in saliva. These results indicate that children with oral cleft are at an increased risk of being infected by S. mutans and lactobacilli at a very early age. Such early colonization indicates a high risk for caries in the primary dentition.
We investigated phosphoenolpyruvate-dependent phosphotransferase system enzyme II activities for sucrose and glucose in Streptococcus mutans 6715g. Two integral membrane proteins, enzyme IIscr and enzyme IIglc, each specific for its sugar substrate, sucrose or glucose, were identified by their abilities to catalyze specific sugar:sugar-phosphate exchange reactions. Some of the properties of these two transport proteins are also presented.
Dodecyl glycerol inhibits the synthesis of the peptidoglycans of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT. This metabolic regulation represents the second known mode by which dodecyl glycerol expresses antibacterial activity. The first mode of action of dodecyl glycerol was shown to stimulate autolysin activity which degrades cell-wall peptidoglycan (Ved HS, Gustow E, Mahadevan V and Pieringer RA, 1984, J. Biol. Chem. 259, 8115-8121).
Growth of Streptococcus mutans Ingbritt on limiting sucrose in a chemostat at dilution rates of 0.05 to 0.4 h-1 (mean generation time, 14 to 1.7 h) resulted in a heterofermentative pattern of metabolic end products. During fast growth, lactic acid was the major end product, whereas at slower growth rates, acetic and formic acids, as well as ethanol, increased to be major end products. The patterns obtained were similar to those seen with the same organism growing on glucose. The glycolytic rate by washed cells was maximum at the lowest dilution rates and decreased as the cells were made to grow faster. Transport of sucrose, glucose, and fructose via the phosphoenolpyruvate phosphotransferase system (PTS) was repressed during growth on sucrose after growth on glucose. Uptake rates suggested that sucrose was transported in the PTS as the intact disaccharide. Comparison of the rate of sugar uptake in the chemostat with the rate of PTS activity in the cells at each growth rate indicated that the PTS was capable of supporting growth only at a dilution rate of 0.05 h-1. Growth on sucrose at faster growth rates required the activity of a second transport system, supporting our earlier observations with glucose that S. mutans contains at least two sugar transport systems.
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.
Many researchers have suggested that the role of glucan-mediated interactions in the adherence of Streptococcus mutans is restricted to accumulation of this cariogenic bacterium following its sucrose (i.e., glucan)-independent binding to saliva-coated tooth surfaces. However, the presence of enzymatically active glucosyltransferase in salivary pellicle suggests that glucans could also promote the initial adherence of S. mutans to the teeth. In the present study, the commonly used hydroxyapatite adherence assay was modified to include the incorporation of glucosyltransferase and the synthesis of glucans in situ on saliva-coated hydroxyapatite beads. Several laboratory strains and clinical isolates of S. mutans were examined for their ability to adhere to experimental pellicles, either with or without the prior formation of glucans in situ. Results showed that most strains of S. mutans bound stereospecifically to glucans synthesized in pellicle. Inhibition studies with various polysaccharides and fungal dextranase indicated that alpha 1,6-linked glucose residues were of primary importance in the glucan binding observed. Scanning electron microscopic analysis showed direct binding of S. mutans to hydroxyapatite surface-associated polysaccharide and revealed no evidence of trapping or cell-to-cell binding. S. mutans strains also attached to host-derived structures in experimental pellicles, and the data suggest that the bacterial adhesins which recognize salivary binding sites were distinct from glucan-binding adhesins. Furthermore, glucans formed in experimental pellicles appeared to mask the host-derived components. These results support the concept that glucans synthesized in salivary pellicle can promote the selective adherence of the cariogenic streptococci which colonize human teeth.
The uptake of U-14C-glucose by resting cells of Streptococcus mutans OMZ-176 was studied in the presence of the artificial sweetener saccharin as well as sodium chloride. Glucose grown cells were resuspended in phosphate buffer (0.05 M, pH 7.8), and the uptake of U-14C-glucose was observed for 150 min in time intervals of 30 min, in the presence of 0.02 and 2.00 mg/ml of sodium saccharin as well as sodium chloride. As compared to the control and the sodium chloride treatments, sodium saccharin at the highest concentration range more than doubled the accumulation of radioactive labelled carbon within the cells.
Rough colonial morphology and bacteriocin production, two properties which may be associated with the cariogenicity of Streptococcus mutans, were transformed into several strain GS-5 mutants defective in each respective property. Transformation was determined by observing the frequency of cotransfer of these properties with different reference markers. The rough colonial transformants were identical to the parental GS-5 strain with respect to ability to synthesize water-insoluble glucans and undergo in vitro sucrose-dependent colonization of glass surfaces. Alterations in the growth medium and the concentration of the initial cell inoculum resulted in an approximate 10-fold increase in the frequency of transformation of strain GS-5 compared to previous observations.
The growth and survival of two strains of Streptococcus mutans in 5% (wt/vol) glucose or sucrose broth was investigated. S. mutans strain C67-1 showed little loss of viability after 30 h of incubation in batch culture in the presence of either sugar. S. mutans strain C67-25, a noncariogenic mutant of C67-1 that has lost the ability of the latter to produce sticky, insoluble extracellular polysaccharide when grown in sucrose broth, showed a dramatic loss of viability after 30 h of incubation in either glucose or sucrose broth, the effect being most marked in the presence of glucose. The loss of viability was shown to be due to acid production. Insoluble extracellular polysaccharide production appears to be a phenomenon favoring the survival of organisms subjected to high sucrose levels. Other factors must be involved, however, since there are differences between the two strains as regards their survival in glucose broth.
The inhibitory effects of NaF, SnF2, and SnCl2 were compared in vitro on the growth of Streptococcus mutans NCTC No. 10449. Sodium fluoride arrested growth at 300 and 600 ppm F (while SnF2 suppressed the rate of growth at 75 ppm F), showed some bactericidal activity at 150 and 300 ppm F, and was totally bactericidal at 600 ppm F. The greater inhibition by SnF2 suggests an additive effect of SN2+. As a measure of a fluoride-free Sn2+ solution, SnCl2 was inappropriate since most of the tin present, at a neutral pH, was Sn4+. The influence of pH was particularly noteworthy with SnF2 at 600 ppm F, where a total bactericidal effect was observed within four h at pH 5.9, while viable cells were still recoverable from four to 40 h with SnF2 at pH 7.2.
Soluble glucan, which was obtained from action of glucosyltransferases (GTFs) of Streptococcus mutans on sucrose, was partially hydrolyzed by acetic acid and examined for human T lymphoblast (MOLT-4) stimulating activity. Addition of the partially hydrolyzed glucan (15-60 microg/ml) stimulated human T cell (39-65%) in a dose dependant manner according to MTT assay. Production of interleukine-2 (IL-2) and interleukine-2 receptor (IL-2R) from T cell was increased by 44.5 and 25%, respectively, by addition of partially hydrolyzed glucan (15 microg/ml). These results indicate that stimulation of human T cells by hydrolyzed glucan is probably caused by its effects on stimulating gene expression of IL-2 and IL-2R of human T cell.
The intact gtfC gene from Streptococcus mutans GS-5 was isolated in Escherichia coli in plasmid vector pUC18. The glucosyltransferase activity expressed by the gene synthesized both low-molecular-weight water-soluble glucan and insoluble glucan in a primer-independent manner. Purification of the enzyme by procedures that minimize proteolytic digestion yielded a purified preparation with a molecular weight of 140,000. Insertional inactivation of the gtfC gene with a streptococcal erythromycin resistance gene fragment followed by transformation of strain GS-5 suggested that the gtfC gene product was required for sucrose-dependent colonization in vitro. In addition, evidence for the presence of a third gtf gene coding for soluble glucan synthesis was obtained following the construction of mutants containing deletions of both the gtfB and gtfC genes.