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1.  Comparative Analysis of Gtf Isozyme Production and Diversity in Isolates of Streptococcus mutans with Different Biofilm Growth Phenotypes 
Journal of Clinical Microbiology  2004;42(10):4586-4592.
Streptococcus mutans is the main pathogenic agent of dental caries. Glucosyltransferases (Gtfs) produced by these bacteria are important virulence factors because they catalyze the extracellular synthesis of glucans that are necessary for bacterial accumulation in the dental biofilm. The diversity of GtfB and GtfC isozymes was analyzed in 44 genotypes of S. mutans that showed a range of abilities to form biofilms in vitro. Several approaches were used to characterize these isozymes, including restriction fragment length polymorphism analysis of the gtfB and gtfC genes, zymographic analysis of the identified GtfB and GtfC genotypes, and quantitation of isozyme production in immunoblot experiments with specific monoclonal antibodies. A high diversity of gtf genes, patterns of enzymatic activity, and isozyme production was identified among the isolates tested. GtfC and, to a lesser extent, GtfB were produced in significantly higher amounts by strains that had high biofilm-forming ability than by strains with low biofilm-forming ability. Biofilm formation was independent of the GtfB and GtfC genotype. Atypical strains that showed an apparent single Gtf isozyme of intermediate size between GtfB and GtfC were also identified. The results indicate that various expression levels of GtfB and GtfC isozymes are associated with the ability of distinct S. mutans genotypes to grow as biofilms, strengthening the results of previous genetic and biochemical studies performed with laboratory strains. These studies also emphasize the need to identify factors that control gtf gene expression.
doi:10.1128/JCM.42.10.4586-4592.2004
PMCID: PMC522304  PMID: 15472313
2.  A Pleiotropic Regulator, Frp, Affects Exopolysaccharide Synthesis, Biofilm Formation, and Competence Development in Streptococcus mutans  
Infection and Immunity  2006;74(8):4581-4589.
Exopolysaccharide synthesis, biofilm formation, and competence are important physiologic functions and virulence factors for Streptococcus mutans. In this study, we report the role of Frp, a transcriptional regulator, on the regulation of these traits crucial to pathogenesis. An Frp-deficient mutant showed decreased transcription of several genes important in virulence, including those encoding fructosyltransferase (Ftf), glucosyltransferase B (GtfB), and GtfC, by reverse transcription and quantitative real-time PCR. Expression of Ftf was decreased in the frp mutant, as assessed by Western blotting as well as by the activity assays. Frp deficiency also inhibited the production of GtfB in the presence of glucose and sucrose as well as the production of GtfC in the presence of glucose. As a consequence of the effects on GtfB and -C, sucrose-induced biofilm formation was decreased in the frp mutant. The expression of competence mediated by the competence-signaling peptide (CSP) system, as assessed by comC gene transcription, was attenuated in the frp mutant. As a result, the transformation efficiency was decreased in the frp mutant but was partially restored by adding synthetic CSP. Transcription of the frp gene was significantly increased in the frp mutant under all conditions tested, indicating that frp transcription is autoregulated. Furthermore, complementation of the frp gene in the frp mutant restored transcription of the affected genes to levels similar to those in the wild-type strain. These results suggest that Frp is a novel pleiotropic effector of multiple cellular functions and is involved in the modulation of exopolysaccharide synthesis, sucrose-dependent biofilm formation, and competence development.
doi:10.1128/IAI.00001-06
PMCID: PMC1539613  PMID: 16861645
3.  Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms▿  
Journal of Bacteriology  2010;192(12):3024-3032.
Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.
doi:10.1128/JB.01649-09
PMCID: PMC2901689  PMID: 20233920
4.  Hydroxy decenoic acid down regulates gtfB and gtfC expression and prevents Streptococcus mutans adherence to the cell surfaces 
Background
10-Hydroxy-2-decenoic acid, an unsaturated fatty acid is the most active and unique component to the royal jelly that has antimicrobial properties. Streptococcus mutans is associated with pathogenesis of oral cavity, gingivoperiodontal diseases and bacteremia following dental manipulations. In the oral cavity, S. mutans colonize the soft tissues including tongue, palate, and buccal mucosa. When considering the role of supragingival dental plaque in caries, the proportion of acid producing bacteria (particularly S. mutans), has direct relevance to the pathogenicity of the plaque. The genes that encode glucosyltransferases (gtfs) especially gtfB and gtfC are important in S. mutans colonization and pathogenesis. This study investigated the hydroxy-decenoic acid (HDA) effects on gtfB and gtfC expression and S. mutans adherence to cells surfaces.
Methods
Streptococcus mutans was treated by different concentrations of HPLC purified HDA supplied by Iran Beekeeping and Veterinary Association. Real time RT-PCR and western blot assays were conducted to evaluate gtfB and gtfC genes transcription and translation before and after HDA treatment. The bacterial attachment to the cell surfaces was evaluated microscopically.
Results
500 μg ml-1 of HDA inhibited gtfB and gtfC mRNA transcription and its expression. The same concentration of HDA decreased 60% the adherence of S. mutans to the surface of P19 cells.
Conclusion
Hydroxy-decenoic acid prevents gtfB and gtfC expression efficiently in the bactericide sub-concentrations and it could effectively reduce S. mutans adherence to the cell surfaces. In the future, therapeutic approaches to affecting S. mutans could be selective and it’s not necessary to put down the oral flora completely.
doi:10.1186/1476-0711-11-21
PMCID: PMC3495742  PMID: 22839724
Biofilm; Caries; Glucosyltransferase; Streptococcus
5.  Influence of Apigenin on gtf Gene Expression in Streptococcus mutans UA159 
Apigenin, a potent inhibitor of glucosyltransferase activity, affects the accumulation of Streptococcus mutans biofilms in vitro by reducing the formation of insoluble glucans and enhancing the soluble glucan content of the polysaccharide matrix. In the present study, we investigated the influence of apigenin on gtfB, gtfC, and gtfD expression in S. mutans UA159. Apigenin (0.1 mM) significantly decreased the expression of gtfB and gtfC mRNA (P < 0.05); in contrast, it increased the expression of gtfD in S. mutans growing in the planktonic state. The protein levels of GTF B, GTF C, and GTF D in culture supernatants were also affected; less GTF B and C were detected, whereas the level of GTF D was significantly elevated (P < 0.05). A similar profile of gtf expression was obtained with biofilms, although an elevated concentration (1 mM) of apigenin was required to elicit the effects. The influence of apigenin on gtf gene expression was independent of any effect on GTF activity, did not involve inhibition of growth or effects on pH, and was not affected by addition of sucrose. The data show that apigenin modulates the genetic expression of virulence factors in S. mutans.
doi:10.1128/AAC.50.2.542-546.2006
PMCID: PMC1366919  PMID: 16436708
6.  A VicRK Signal Transduction System in Streptococcus mutans Affects gtfBCD, gbpB, and ftf Expression, Biofilm Formation, and Genetic Competence Development 
Journal of Bacteriology  2005;187(12):4064-4076.
Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic+). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic+. Based on real-time PCR, Smuvic+ showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in S. mutans.
doi:10.1128/JB.187.12.4064-4076.2005
PMCID: PMC1151735  PMID: 15937169
7.  Production, characterization, and application of monoclonal antibodies which distinguish three glucosyltransferases from Streptococcus mutans. 
Infection and Immunity  1993;61(1):323-328.
Thirty-three murine monoclonal antibodies (MAbs) against the three glucosyltransferases (GTFs) (GTF-I, -SI, and -S) from Streptococcus mutans were obtained by the fusion of murine myeloma cells (P3X63-Ag8-U1) with spleen cells of BALB/c mice immunized with pure GTF-S or partially purified GTF-I from serotype c S. mutans PS14. The immunoreactivities of these MAbs were tested by enzyme-linked immunosorbent assay and Western blotting (immunoblotting) with various GTF preparations. GTF-I and GTF-SI were expressed from two Streptococcus milleri or Escherichia coli transformants harboring gtfB or gtfC, respectively. All of the five MAbs raised against the GTF-S from PS14 reacted only with the homologous enzymes. Of these, 8 MAbs reacted only with the gtfB gene product (GTF-I), 4 MAbs reacted only with the gtfC gene product (GTF-SI), and the remaining 16 MAbs reacted with both gene products. The existence of GTF-SI in the purified GTF-I from PS14 was demonstrated by Western blot analysis using the representative monospecific MAbs. Further, the relative levels of the three GTFs in the extracellular and cellular fractions of S. mutans clinical isolates were examined by immunoblot analysis. The findings indicated that the relative level of GTF-SI, unlike that of GTF-I or GTF-S, differed markedly among isolates although the three GTFs were synthesized extracellularly by all the strains.
Images
PMCID: PMC302722  PMID: 8418055
8.  α-Mangostin Disrupts the Development of Streptococcus mutans Biofilms and Facilitates Its Mechanical Removal 
PLoS ONE  2014;9(10):e111312.
α-Mangostin (αMG) has been reported to be an effective antimicrobial agent against planktonic cells of Streptococcus mutans, a biofilm-forming and acid-producing cariogenic organism. However, its anti-biofilm activity remains to be determined. We examined whether αMG, a xanthone purified from Garcinia mangostana L grown in Vietnam, disrupts the development, acidogenicity, and/or the mechanical stability of S. mutans biofilms. Treatment regimens simulating those experienced clinically (twice-daily, 60 s exposure each) were used to assess the bioactivity of αMG using a saliva-coated hydroxyapatite (sHA) biofilm model. Topical applications of early-formed biofilms with αMG (150 µM) effectively reduced further biomass accumulation and disrupted the 3D architecture of S. mutans biofilms. Biofilms treated with αMG had lower amounts of extracellular insoluble and intracellular iodophilic polysaccharides (30–45%) than those treated with vehicle control (P<0.05), while the number of viable bacterial counts was unaffected. Furthermore, αMG treatments significantly compromised the mechanical stability of the biofilm, facilitating its removal from the sHA surface when subjected to a constant shear stress of 0.809 N/m2 (>3-fold biofilm detachment from sHA vs. vehicle-treated biofilms; P<0.05). Moreover, acid production by S. mutans biofilms was disrupted following αMG treatments (vs. vehicle-control, P<0.05). The activity of enzymes associated with glucan synthesis, acid production, and acid tolerance (glucosyltransferases B and C, phosphotransferase-PTS system, and F1F0-ATPase) were significantly inhibited by αMG. The expression of manL, encoding a key component of the mannose PTS, and gtfB were slightly repressed by αMG treatment (P<0.05), while the expression of atpD (encoding F-ATPase) and gtfC genes was unaffected. Hence, this study reveals that brief exposures to αMG can disrupt the development and structural integrity of S. mutans biofilms, at least in part via inhibition of key enzymatic systems associated with exopolysaccharide synthesis and acidogenicity. αMG could be an effective anti-virulence additive for the control and/or removal of cariogenic biofilms.
doi:10.1371/journal.pone.0111312
PMCID: PMC4211880  PMID: 25350668
9.  Cariogenicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by allelic exchange. 
Infection and Immunity  1991;59(7):2316-2323.
Streptococcus mutans produces several enzymes which metabolize sucrose. Three glucosyltransferase genes (gtfB, gtfC, and gtfD) and a single fructosyltransferase gene (ftf) encode enzymes which are important in formation of exopolysaccharides. Mutants of S. mutans V403 carrying single and multiple mutations of the gtfB, gtfC, gtfD, and ftf genes recently have been constructed by allelic exchange in our laboratory. Using selected strains from this panel of mutants, we examined the importance of water-insoluble glucan, water-soluble glucan, and fructan production in cariogenicity while controlling for the effects of strain and species variability. Genetic and biochemical characterization of mutants and assays of glucosyltransferase and fructosyltransferase activities were performed to ensure that the phenotypes of strains coincided with deficiencies predicted by genotype. The young gnotobiotic rat model of cariogenicity was used to assess virulence of the wild-type strain and isogenic mutants. Mutant strains were less virulent than the wild type in almost every location examined for caries on tooth surfaces and level of involvement of lesions (depth and severity). Inactivation of either gtfB and gtfC or ftf dramatically reduced virulence; the subsequent inactivation of gtfD did not enhance the effect of reduced virulence.
Images
PMCID: PMC258013  PMID: 1828790
10.  Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependent soluble glucan synthesis. 
Infection and Immunity  1989;57(7):2079-2085.
Two glucosyltransferase genes from Streptococcus mutans GS-5, gtfB and gtfC, have been previously isolated and sequenced in this laboratory. In the present communication a third gtf gene, gtfD, was isolated and characterized. Isolation of the gene involved a novel procedure utilizing the integration plasmid pVA891. A peptide expressed by the 1.7-kilobase DNA fragment from strain NHS1 (containing deletions in both the gtfB and gtfC genes) was initially identified in a pUC18 clone bank with antiglucosyltransferase antibodies. This fragment was integrated into the GS-5 chromosome following ligation into pVA891 and transformation, yielding strain DP2. The vector together with one complete and one incomplete copy of the gtfD gene was removed from the chromosome of strain DP2 following EcoRI digestion, religation, and transformation of E. coli HB101. The resultant plasmid, pNH4, expressed glucosyltransferase S (GTF-S) activity. The enzyme was purified to near homogeneity and was shown to synthesize water-soluble glucan exclusively in a primer-dependent manner. The molecular mass (155 kilodaltons) and the kinetic parameters of the purified enzyme were similar to those observed for the GTF-S enzyme previously purified from culture fluids of strain GS-5. Insertional inactivation of the gtfD gene indicated that this gene is not required for in vitro sucrose-dependent adherence to smooth surfaces. Furthermore, inactivation of the gtfD gene in a gtfC gtfB mutant indicated that three distinct gtf genes involved in glucan formation are present on the S. mutans GS-5 chromosome. Southern blot analysis further suggested that the gtfD gene does not share demonstrable homology with the gtf genes from Streptococcus sanguis or Streptococcus sobrinus.
Images
PMCID: PMC313844  PMID: 2543630
11.  The Streptococcus mutans vicX Gene Product Modulates gtfB/C Expression, Biofilm Formation, Genetic Competence, and Oxidative Stress Tolerance▿  
Journal of Bacteriology  2006;189(4):1451-1458.
Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity.
doi:10.1128/JB.01161-06
PMCID: PMC1797355  PMID: 17114248
12.  Expression of Streptococcus mutans gtf genes in Streptococcus milleri. 
Infection and Immunity  1992;60(7):2815-2822.
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.
Images
PMCID: PMC257239  PMID: 1377183
13.  The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm 
PLoS Pathogens  2012;8(4):e1002623.
Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.
Author Summary
Virulent biofilms formed on surfaces are associated with many human infections. The disease dental caries, expressed as cavities, is a prime example of the consequences arising from interactions between bacteria and sugars on tooth-surfaces. When Streptococcus mutans metabolize sugars, they produce a glue-like polymer termed glucan, helping them to adhere firmly to teeth. Glucan is also formed on bacterial surfaces in the mouth, and will accumulate and enmesh additional microorganisms creating the gelatinous formation known as dental plaque-biofilm. We found unique islets of bacteria within these biofilms, particularly close to the tooth-surface, providing safe havens in which bacteria thrive and produce acids that erode teeth. One intriguing mystery is why acids accumulate on the tooth-surface when there is an abundance of neutral-pH saliva surrounding the teeth. We found that bacterial-islets are particularly protected by glucan, which retards neutralization. We noticed that, within biofilms, the interiors of these islets are acidic, where only acid-tolerant bacteria can prosper, ensuring continued localized acid production. Our study demonstrates that construction of biofilms mediated by glucans forms complex 3D architectures, creating a variety of acidic-microenvironments that are essential for virulence expression. These results may aid in the development of enhanced methods to modulate biofilm formation.
doi:10.1371/journal.ppat.1002623
PMCID: PMC3320608  PMID: 22496649
14.  Functional Analyses of a Conserved Region in Glucosyltransferases of Streptococcus mutans 
Infection and Immunity  1998;66(10):4797-4803.
Streptococcus mutans glucosyltransferases (GTFs; GtfB, -C, and -D) synthesize water-soluble and -insoluble glucan polymers from sucrose. We have identified previously a conserved region of 19 amino acids (aa) (Gtf-P1; aa 409 to 427 of GtfB and aa 435 to 453 of GtfC) which is functionally important for both enzymatic activity and bacterial adherence. Monoclonal antibodies directed against Gtf-P1 selectively inhibited insoluble glucan synthesis by GtfB and -C but had no effect on soluble glucan synthesis by GtfD, suggesting that despite an apparent near identity of sequence, corresponding residues may function differently in these enzymes. To test this hypothesis, we used different strategies of mutagenesis to analyze amino acid residues of GtfB and GtfC in Gtf-P1. In-frame insertion of 6 amino acids preceding, or deletion of 14 amino acids within, this conserved region abolished the enzymatic activities of both GtfB and GtfC. Substitution of several residues in combination by random mutagenesis resulted in GtfB, but not GtfC, enzymes exhibiting decreased glucan synthesis and reduced rates of sucrose hydrolysis. Amino acid substitutions of Asp residues in GtfB or GtfC were found to be more critical for enzymatic activity than at other positions of this region. Interestingly, single mutation at Asp411 or Asp413 of GtfB resulted in enzymes retaining about 20% of wild-type activity, whereas mutagenesis of the corresponding Asp at position 437 or 439 in GtfC resulted in complete loss of enzymatic activity. Furthermore, single amino acid substitution of a Val residue between the two Asp residues enhanced the sucrase- and glucan-synthesizing activities of GtfB and GtfC. These results confirmed the report from another laboratory that Asp residues in the Gtf-P1 region are essential for enzymatic catalysis and provide new evidence that identical residues may function differently in closely related Gtf enzymes.
PMCID: PMC108592  PMID: 9746581
15.  Phylogenetic Analysis of Glucosyltransferases and Implications for the Coevolution of Mutans Streptococci with Their Mammalian Hosts 
PLoS ONE  2013;8(2):e56305.
Glucosyltransferases (Gtfs) catalyze the synthesis of glucans from sucrose and are produced by several species of lactic-acid bacteria. The oral bacterium Streptococcus mutans produces large amounts of glucans through the action of three Gtfs. GtfD produces water-soluble glucan (WSG), GtfB synthesizes water-insoluble glucans (WIG) and GtfC produces mainly WIG but also WSG. These enzymes, especially those synthesizing WIG, are of particular interest because of their role in the formation of dental plaque, an environment where S. mutans can thrive and produce lactic acid, promoting the formation of dental caries. We sequenced the gtfB, gtfC and gtfD genes from several mutans streptococcal strains isolated from the oral cavity of humans and searched for their homologues in strains isolated from chimpanzees and macaque monkeys. The sequence data were analyzed in conjunction with the available Gtf sequences from other bacteria in the genera Streptococcus, Lactobacillus and Leuconostoc to gain insights into the evolutionary history of this family of enzymes, with a particular emphasis on S. mutans Gtfs. Our analyses indicate that streptococcal Gtfs arose from a common ancestral progenitor gene, and that they expanded to form two clades according to the type of glucan they synthesize. We also show that the clade of streptococcal Gtfs synthesizing WIG appeared shortly after the divergence of viviparous, dentate mammals, which potentially contributed to the formation of dental plaque and the establishment of several streptococci in the oral cavity. The two S. mutans Gtfs capable of WIG synthesis, GtfB and GtfC, are likely the product of a gene duplication event. We dated this event to coincide with the divergence of the genomes of ancestral early primates. Thus, the acquisition and diversification of S. mutans Gtfs predates modern humans and is unrelated to the increase in dietary sucrose consumption.
doi:10.1371/journal.pone.0056305
PMCID: PMC3572963  PMID: 23457545
16.  Manganese Affects Streptococcus mutans Virulence Gene Expression 
Caries Research  2007;41(6):503-511.
Background/Aims
Studies of trace metals in drinking water and tooth enamel have suggested a caries-promoting potential for manganese (Mn). Additionally, Mn has been shown to be essential for the expression of mutans streptococci virulence factors such as the glucan-binding lectin (GBL) of Streptococcus sobrinus. The Streptococcus mutans glucan-binding protein (Gbp) GbpC is the functional analogue of the S. sobrinus GBL. S. mutans Gbps have been shown to contribute to biofilm architecture and virulence. This study was undertaken to examine the effects of Mn on the transcription of genes encoding S. mutans Gbps, including gbpC, along with other critical S. mutans virulence genes.
Methods
Microarray analyses suggested the potential for an Mn effect on Gbp genes. Further investigation of the Mn effects on selected genes was undertaken by performing Northern blots, Western blots, and RT-PCR under conditions of planktonic and biofilm growth in Mn-depleted media or in media containing 50 μM Mn.
Results
Mn resulted in increased expression of gbpC and gtfB, and decreased expression of wapA, in both planktonic and biofilm cultures. The expression levels of gbpA and gbpD were also decreased in the presence of Mn, but only in biofilms. The expression of gtfC was increased in the presence of Mn only in planktonic cultures. The spaP gene was expressed more highly in Mn-supplemented planktonic cultures but less in Mn-supplemented biofilms.
Conclusion
Mn availability affects the expression of multiple S. mutans genes involved in adhesion and biofilm formation. Furthermore, these effects depend on the growth state of the organism.
doi:10.1159/000110883
PMCID: PMC2820327  PMID: 17992013
Biofilm; Manganese; Streptococcus mutans virulence
17.  Biology of Streptococcus mutans-Derived Glucosyltransferases: Role in Extracellular Matrix Formation of Cariogenic Biofilms 
Caries Research  2011;45(1):69-86.
The importance of Streptococcus mutans in the etiology and pathogenesis of dental caries is certainly controversial, in part because excessive attention is paid to the numbers of S. mutans and acid production while the matrix within dental plaque has been neglected. S. mutans does not always dominate within plaque; many organisms are equally acidogenic and aciduric. It is also recognized that glucosyltransferases from S. mutans (Gtfs) play critical roles in the development of virulent dental plaque. Gtfs adsorb to enamel synthesizing glucans in situ, providing sites for avid colonization by microorganisms and an insoluble matrix for plaque. Gtfs also adsorb to surfaces of other oral microorganisms converting them to glucan producers. S. mutans expresses 3 genetically distinct Gtfs; each appears to play a different but overlapping role in the formation of virulent plaque. GtfC is adsorbed to enamel within pellicle whereas GtfB binds avidly to bacteria promoting tight cell clustering, and enhancing cohesion of plaque. GtfD forms a soluble, readily metabolizable polysaccharide and acts as a primer for GtfB. The behavior of soluble Gtfs does not mirror that observed with surface-adsorbed enzymes. Furthermore, the structure of polysaccharide matrix changes over time as a result of the action of mutanases and dextranases within plaque. Gtfs at distinct loci offer chemotherapeutic targets to prevent caries. Nevertheless, agents that inhibit Gtfs in solution frequently have a reduced or no effect on adsorbed enzymes. Clearly, conformational changes and reactions of Gtfs on surfaces are complex and modulate the pathogenesis of dental caries in situ, deserving further investigation.
doi:10.1159/000324598
PMCID: PMC3068567  PMID: 21346355
Biofilms; Dental caries; Extracellular matrix; Glucosyltransferases; Polysaccharides; Streptococcus mutans
18.  Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms 
BMC Microbiology  2009;9:228.
Background
The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.
Results
Twice-daily treatment (one-minute exposure) during biofilm formation affected the gene expression by S. mutans both at early (49-h) and later (97-h) stages of biofilm development. Biofilms treated with combination of agents displayed lower mRNA levels for gtfB and gtfD (associated with exopolysaccharides synthesis) and aguD (associated with S. mutans acid tolerance) than those treated with vehicle-control (p < 0.05). Furthermore, treatment with combination of agents markedly affected the structure-architecture of S. mutans biofilms by reducing the biovolume (biomass) and proportions of both EPS and bacterial cells across the biofilm depth, especially in the middle and outer layers (vs. vehicle-control, p < 0.05). The biofilms treated with combination of agents were also less acidogenic, and had reduced amounts of extracellular insoluble glucans and intracellular polysaccharides than vehicle-treated biofilms (p < 0.05).
Conclusion
The data show that the combination of naturally-occurring agents with fluoride effectively disrupted the expression of specific virulence genes, structural organization and accumulation of S. mutans biofilms, which may explain the enhanced cariostatic effect of our chemotherapeutic approach.
doi:10.1186/1471-2180-9-228
PMCID: PMC2774857  PMID: 19863808
19.  Regulation of the Glucosyltransferase (gtfBC) Operon by CovR in Streptococcus mutans 
Journal of Bacteriology  2006;188(3):988-998.
Streptococcus mutans is an important etiological agent of dental caries in humans. The extracellular polysaccharides synthesized by cell-associated glucosyltransferases (encoded by gtfBC) from sucrose have been recognized as one of the important virulence factors that promote cell aggregation and adherence to teeth, leading to dental plaque formation. In this study, we have characterized the effect of CovR, a global response regulator, on glucosyltransferase expression. Inactivation of covR in strain UA159 resulted in a marked increase in the GtfB and GtfC proteins, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With the use of a transcriptional reporter system of a single chromosomal copy of the PgtfB-gusA and PgtfC-gusA fusions, we confirmed the transcriptional regulation of these promoters by CovR. By in vitro electrophoretic mobility shift assays with purified CovR protein, we showed that CovR regulates these promoters directly. DNase I footprinting analyses suggest that CovR binds to large regions on these promoters near the transcription start sites. Taken together, our results indicate that CovR negatively regulates the expression of the gtfB and gtfC genes by directly binding to the promoter region.
doi:10.1128/JB.188.3.988-998.2006
PMCID: PMC1347363  PMID: 16428403
20.  Comparison of quaternary ammonium-containing with nano-silver-containing adhesive in antibacterial properties and cytotoxicity 
Objective
Antibacterial primer and adhesive are promising to help combat biofilms and recurrent caries. The objectives of this study were to compare novel bonding agent containing quaternary ammonium dimethacrylate (QADM) with bonding agent containing nanoparticles of silver (NAg) in antibacterial activity, contact-inhibition vs. long-distance inhibition, glucosyltransferases (gtf) gene expressions, and cytotoxicity for the first time.
Methods
QADM and NAg were incorporated into Scotchbond Multi-Purpose adhesive and primer. Microtensile dentin bond strength was measured. Streptococcus mutans (S. mutans) biofilm on resin surface (contact-inhibition) as well as S. mutans in culture medium away from the resin surface (long-distance inhibition) were tested for metabolic activity, colony-forming units (CFU), lactic acid production, and gtf gene expressions. Eluents from cured primer/adhesive samples were used to examine cytotoxicity against human gingival fibroblasts.
Results
Bonding agent with QADM greatly reduced CFU and lactic acid of biofilms on the resin surface (p < 0.05), while having no effect on S. mutans in culture medium away from the resin surface. In contrast, bonding agent with NAg inhibited not only S. mutans on the resin surface, but also S. mutans in culture medium away from the resin surface. Bonding agent with QADM suppressed gtfB, gtfC and gtfD gene expressions of S. mutans on its surface, but not away from its surface. Bonding agent with NAg suppressed S. mutans gene expressions both on its surface and away from its surface. Bonding agents with QADM and NAg did not adversely affect microtensile bond strength or fibroblast cytotoxicity, compared to control (p > 0.1).
Significance
QADM-containing adhesive had contact-inhibition and inhibited bacteria on its surface, but not away from its surface. NAg-containing adhesive had long-distance killing capability and inhibited bacteria on its surface and away from its surface. The novel antibacterial adhesives are promising for caries-inhibition restorations, and QADM and NAg could be complimentary agents in inhibiting bacteria on resin surface as well as away from resin surface.
doi:10.1016/j.dental.2013.01.012
PMCID: PMC3631003  PMID: 23428077
Antibacterial dental adhesive; quaternary ammonium; silver nanoparticles; Streptococcus mutans biofilm; microtensile dentin bond strength; fibroblast cytotoxicity
21.  Real-Time Monitoring of Streptococcus mutans Biofilm Formation Using a Quartz Crystal Microbalance 
Caries Research  2007;41(6):474-483.
The ability of Streptococcus mutans, a well-known etiological agent in dental caries, to attach and form a biofilm is an important key to its virulence. The effects of various environmental factors (i.e. sucrose concentration, flow rate and temperature as well as genetic manipulations) on the capability of S. mutans (UA 140) to attach, form and detach were monitored in situ using quartz crystal microbalance. The biofilm growth rate was much slower than that of planktonic growth. Greater availability of sucrose contributed to biofilms with less lag time, lower doubling times and earlier detachment. Flow rate experiments showed that as the shear stress was reduced, the maximum mass accumulated also decreased. However, the detachment process was independent of shear force, perhaps indicative of quorum sensing. Increasing the incubation temperature from 37 to 40°C extended the lag period and inhibited the ability of the biofilm to attach readily. Absence of either the ciaH, luxS, gtfB or gtfC genes also greatly affected the ability of the S. mutans to adhere to a surface in comparison to the wild type. Quartz crystal microbalance results indicate that the gtfC gene possibly has a greater contribution to biofilm attachment than the gtfB gene, that the presence of the luxS gene is critical for attachment and that the ciaH gene primarily affects the initial reversible attachment of the biofilm.
doi:10.1159/000108321
PMCID: PMC2820325  PMID: 17851235
In situ; Mutants; Quartz crystal microbalance; Real-time; Streptococcus mutans
22.  Molecular cloning and characterization of the glucosyltransferase C gene (gtfC) from Streptococcus mutans LM7. 
Infection and Immunity  1987;55(9):2176-2182.
A glucosyltransferase (GTF) gene, designated gtfC, was cloned from Streptococcus mutans LM7. Its gene product was detected by screening a bacteriophage lambda library with rabbit antiserum raised against S. mutans LM7 extracellular proteins. DNA isolated from the immunopositive recombinant phage revealed two S. mutans chromosomal EcoRI fragment inserts, 8.1 and 4.7 kilobase pairs in size. Escherichia coli minicell analyses revealed the approximate position and direction of transcription of the gtfC gene. The gene product was determined to be a polypeptide of about 150 kilodaltons which synthesized a water-soluble glucan. Restriction endonuclease mapping and DNA hybridization indicated a repeated region of DNA corresponding to a portion of the coding region of gtfC immediately downstream from the intact gtfC locus on the chromosome. A 300-base-pair gtfC-specific probe showed that the gene and the putative duplicated sequence were present in S. mutans serotypes c, e, and f, but not in other related oral streptococci which had GTF activity. In addition, the gtfC determinant displayed homology to sequences corresponding to the carboxy-terminal coding region of a gene (gtfB) encoding a GTF activity that synthesized water-insoluble glucans. These data suggest that at least one class of GTF genes may be present in multiple copies in S. mutans and, further, that GTF genes may contain conserved sequences internal to their coding regions.
Images
PMCID: PMC260675  PMID: 3040591
23.  Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo 
Infection and Immunity  2014;82(5):1968-1981.
Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.
doi:10.1128/IAI.00087-14
PMCID: PMC3993459  PMID: 24566629
24.  Lactobacillus Acidophilus-Derived Biosurfactant Effect on GTFB and GTFC Expression Level in Streptococcus Mutans Biofilm Cells 
Brazilian Journal of Microbiology  2011;42(1):330-339.
Streptococcus mutans (S. mutans), harboring biofilm formation, considered as a main aetiological factor of dental caries. Gtf genes play an important role in S. mutans biofilm formation. The purpose of this study was to investigate the effect of Lactobacillus acidophilus–derived biosurfactant on S. mutans biofilm formation and gtfB/C expression level (S. mutans standard strain ATCC35668 and isolated S. mutans strain (22) from dental plaque).
The Lactobacillus acidophilus (L. acidophilus) DSM 20079 was selected as a probiotic strain to produce biosurfactant. The FTIR analysis of its biosurfactant showed that it appears to have a protein-like component. Due to the release of such biosurfactants, L. acidophilus was able to interfere in the adhesion and biofilm formation of the S. mutans to glass slide. It also could make streptococcal chains shorter. Using realtime RT-PCR quantitation method made it clear that gtfB and gtfC gene expression were decreased in the presence of L. acidophilus–derived biosurfactant fraction.
Several properties of S. mutans cells (the surface properties, biofilm formation, adhesion ability and gene expression) were changed after L. acidophilus- derived biosurfactant treatment. It is also concluded that biosurfacant treatment can provide an optional way to control biofilm development. On the basis of our findings, we can suggest that the prepared biosurfactant may interfere with adhesion processes of S. mutans to teeth surfaces, provided additional evaluation produce satisfactory results.
doi:10.1590/S1517-83822011000100042
PMCID: PMC3768947  PMID: 24031639
Biofilm formation; biosurfactant; gtfB/C; Lactobacillus acidophilus; real-time RT PCR; Streptococcus mutans
25.  Inactivation of the gbpA Gene of Streptococcus mutans Alters Structural and Functional Aspects of Plaque Biofilm Which Are Compensated by Recombination of the gtfB and gtfC Genes 
Infection and Immunity  1999;67(8):3909-3914.
Inactivation of the gbpA gene of Streptococcus mutans increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which gtfB and gtfC have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180–2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters S. mutans plaque structure and that the presence of gtfBC recombinant organisms within a gbpA background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt S. mutans plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the gbpA mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the gbpA mutants used as inoculum were also gtfBC recombinants (gbpA/25%gtfBC), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin; gbpA plaque organisms were more susceptible than organisms in either the wt or gbpA/25%gtfBC plaques. These data allow the conclusion that GbpA contributes to S. mutans plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that S. mutans biofilm structure influences virulence. A potential model for this influence, which can account for the gtfBC recombination compensating gbpA inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development.
PMCID: PMC96671  PMID: 10417155

Results 1-25 (912157)