d‐Alanine metabolism is essential for growth and biofilm formation of Streptococcus mutans

Part of the d ‐alanine (d ‐Ala) metabolic pathway in bacteria involves the conversion of l ‐alanine to d ‐Ala by alanine racemase and the formation of d ‐alanyl–d ‐alanine by d ‐alanine–d ‐alanine ligase, the product of which is involved in cell wall peptidoglycan synthesis. At present, drugs that target the metabolic pathway of d ‐Ala are already in clinical use – e.g. d ‐cycloserine (DCS) is used as an antibiotic against Mycobacterium tuberculosis. Streptococcus mutans is the main cariogenic bacterium in the oral cavity. Its d ‐Ala metabolism‐associated enzymes alanine racemase and d ‐alanine–d ‐alanine ligase are encoded by the genes smu.1834 and smu.599, respectively, which may be potential targets for inhibitors. In this study, the addition of DCS blocked the d ‐Ala metabolic pathway in S. mutans, leading to bacterial cell wall defects, significant inhibition of bacterial growth and biofilm formation, and reductions in extracellular polysaccharide production and bacterial adhesion. However, the exogenous addition of d ‐Ala could reverse the inhibitory effect of DCS. Through the means of drug regulation, our study demonstrated, for the first time, the importance of d ‐Ala metabolism in the survival and biofilm formation of S. mutans. If the growth of S. mutans can be specifically inhibited by designing drugs that target d ‐Ala metabolism, then this may serve as a potential new treatment for dental caries.     

Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism,acid tolerance response and biofilm formation

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA‐binding N‐terminal domain and a C‐terminal domain highly homologous to the pyridoxal phosphate‐dependent aspartate aminotransferases. A pdxR‐deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR‐deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real‐time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B₆ biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR‐deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B₆. Further studies revealed that although S. mutans is known to require vitamin B₆ to grow in defined medium, B₆ vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B₆ metabolism, acid tolerance response and biofilm formation.     

In silico analysis of the competition between Streptococcus sanguinis and Streptococcus mutans in the dental biofilm

During dental caries, the dental biofilm modifies the composition of the hundreds of involved bacterial species. Changing environmental conditions influence competition. A pertinent model to exemplify the complex interplay of the microorganisms in the human dental biofilm is the competition between Streptococcus sanguinis and Streptococcus mutans. It has been reported that children and adults harbor greater numbers of S. sanguinis in the oral cavity, associated with caries‐free teeth. Conversely, S. mutans is predominant in individuals with a high number of carious lesions. Competition between both microorganisms stems from the production of H₂O₂ by S. sanguinis and mutacins, a type of bacteriocins, by S. mutans. There is limited evidence on how S. sanguinis survives its own H₂O₂ levels, or if it has other mechanisms that might aid in the competition against S. mutans, nonetheless. We performed a genomic and metabolic pathway comparison, coupled with a comprehensive literature review, to better understand the competition between these two species. Results indicated that S. sanguinis can outcompete S. mutans by the production of an enzyme capable of metabolizing H₂O₂. S. mutans, however, lacks the enzyme and is susceptible to the peroxide from S. sanguinis. In addition, S. sanguinis can generate energy through gluconeogenesis and seems to have evolved different communication mechanisms, indicating that novel proteins may be responsible for intra‐species communication.     

EzrA,a cell shape regulator contributing to biofilm formation and competitiveness in Streptococcus mutans

Bacterial cell division is initiated by tubulin homologue FtsZ that assembles into a ring structure at mid‐cell to facilitate cytokinesis. EzrA has been identified to be implicated in FtsZ‐ring dynamics and cell wall biosynthesis during cell division of Bacillus subtilis and Staphylococcus aureus, the model rod and cocci. However, its role in pathogenic streptococci remains largely unknown. Here, the role of EzrA was investigated in Streptococcus mutans, the primary etiological agent of human dental caries, by constructing an ezrA in‐frame deletion mutant. Our data showed that the ezrA mutant was slow‐growing with a shortened length and extended width round cell shape compared to the wild type, indicating a delay in cell division with abnormalities of peptidoglycan biosynthesis. Additionally, FtsZ irregularly localized in dividing ezrA mutant cells forming angled division planes, potentially contributing to an aberrant cell shape. Furthermore, investigation using single‐species cariogenic biofilm model revealed that deletion of ezrA resulted in defective biofilm formation with less extracellular polysaccharides and altered three‐dimensional biofilm architecture. Unexpectedly, in a dual‐species ecological model, the ezrA mutant exhibited substantially lower tolerance for H₂O₂ and reduced competitiveness against one commensal species, Streptococcus sanguinis. Taken together, these results demonstrate that EzrA plays a key role in regulating cell division and maintaining a normal morphology in S. mutans and is required for its robust biofilm formation/interspecies competition. Therefore, EzrA protein represents a potential therapeutic target in the development of drugs controlling dental caries and other biofilm‐related diseases.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

Mutacin production in Streptococcus mutans genotypes isolated from caries-affected and caries-free individuals

Relationships between genetic diversity and mutacin production in Streptococcus mutans were evaluated in 319 clinical isolates from eight caries-affected and eight caries-free individuals. The isolates were submitted to mutacin typing and AP-PCR (arbitrarily primed polymerase chain reaction) assay. The mutacin production was detected for 12 Streptococcus sp. indicator strains. Results showed significant variations in the mutacin production profiles and the inhibitory spectra of both groups. A possible association was seen between mutacin activity and the distinct patterns of Streptococcus sp. colonization in the two groups. Genotyping by AP-PCR using the primers OPA-02 and OPA-13 revealed 101 distinct genotypes against 48 phenotypes identified by mutacin typing. No correlation was observed between the inhibitory spectra of mutacin and genotypic similarities based on AP-PCR analyses. According to our results, strains of the same S. mutans genotype showed different mutacin profiles, suggesting a high degree of interstrain diversity. In conclusion, mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species.     

Effects of the natural compound,oxyresveratrol, on the growth of Streptococcus mutans,and on biofilm formation,acid production,and virulence gene expression

Streptococcus mutans is one of the major pathogens of dental caries. Oxyresveratrol, a natural compound found in plants, exerts inhibitory effects on many bacterial species but its effect on S. mutans is unknown. The objective of this study was to clarify the antibacterial effect of oxyresveratrol on S. mutans, including effects on basic viability, acidogenicity, acidurity, and extracellular polysaccharide synthesis. The expression of nine genes that encode virulence and protective factors in S. mutans was measured by qRT-PCR. Oxyresveratrol showed a dose-dependent inhibitory effect on survival of S. mutans. At 250 μg ml⁻¹, oxyresveratrol reduced the S. mutans survival rate, inhibited synthesis of water-insoluble glucans, compromised biofilm formation, and significantly down-regulated the expression of glucosyltransferase-I (gtfB) and glucosyltransferase-SI (gtfC). However, the enzymatic activity of lactate dehydrogenase protein was increased and the expression of lactate dehydrogenase (ldh) and ATP synthase subunit beta (atpD) genes were also up-regulated. Besides, glucosyltransferase S (gtfD) up-regulation indicated that water-soluble glucan synthesis was promoted. The vicR, liaR, and comDE genes, which exert a self-protective function in response to external stress, were also up-regulated. In conclusion, oxyresveratrol inhibited the growth of S. mutans and also reduced biofilm formation, acid production, and synthesis of water-insoluble glucans by this organism. In addition, oxyresveratrol also activated a series of S. mutans self-protection mechanisms.     

变异链球菌葡聚糖结合蛋白B的生物学特性研究进展

变异链球菌是人类龋病的主要致病菌,葡聚糖结合蛋白是其重要的毒力因子之一.葡聚糖结合蛋白B在变异链球菌的致龋、维持细菌形态、发挥细胞壁生理功能等方面有重要作用,同时其N端具有免疫原性,可应用于免疫防龋.下面就葡聚糖结合蛋白B的生物学特性研究进展作一综述.     

Deficiency of BrpA in Streptococcus mutans reduces virulence in rat caries model

Our recent studies have shown that BrpA in Streptococcus mutans plays a critical role in cell envelope biogenesis, stress responses, and biofilm formation. In this study, a 10‐species consortium was used to assess how BrpA deficiency influences the establishment, persistence, and competitiveness of S. mutans during growth in a community under conditions typical of the oral cavity. Results showed that, like the wild‐type, the brpA mutant was able to colonize and establish on the surfaces tested. Relative to the wild‐type, however, the brpA mutant had a reduced ability to persist and grow in the 10‐species consortium (P < .001). A rat caries model was also used to examine the effect of BrpA, as well as Psr, a BrpA paralog, on S. mutans cariogenicity. The results showed no major differences in infectivity between the wild‐type and the brpA and psr mutants. Unlike the wild‐type, however, infection with the brpA mutant, but not the psr mutant, showed no significant differences in both total numbers of carious lesions and caries severity, compared with the control group that received bacterial growth medium (P > .05). Metagenomic and quantitative polymerase chain reaction analysis showed that S. mutans infection caused major alterations in the composition of the rats’ plaque microbiota and that significantly less S. mutans was identified in the rats infected with the brpA mutant compared with those infected with the wild‐type and the psr mutant. These results further suggest that BrpA plays a critical role in S. mutans pathophysiology and that BrpA has potential as a therapeutic target in the modulation of S. mutans virulence.     

Mutacin production in Streptococcus mutans genotypes isolated from caries‐affected and caries‐free individuals

Relationships between genetic diversity and mutacin production in Streptococcus mutans were evaluated in 319 clinical isolates from eight caries‐affected and eight caries‐free individuals. The isolates were submitted to mutacin typing and AP‐PCR (arbitrarily primed polymerase chain reaction) assay. The mutacin production was detected for 12 Streptococcus sp. indicator strains. Results showed significant variations in the mutacin production profiles and the inhibitory spectra of both groups. A possible association was seen between mutacin activity and the distinct patterns of Streptococcus sp. colonization in the two groups. Genotyping by AP‐PCR using the primers OPA‐02 and OPA‐13 revealed 101 distinct genotypes against 48 phenotypes identified by mutacin typing. No correlation was observed between the inhibitory spectra of mutacin and genotypic similarities based on AP‐PCR analyses. According to our results, strains of the same S. mutans genotype showed different mutacin profiles, suggesting a high degree of interstrain diversity. In conclusion, mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species.     

Isolation and characterization of Lactobacillus species inhibiting the formation of Streptococcus mutans biofilm

Oral bacterium S11 was isolated from the saliva of young children without dental caries and with little or no visible supragingival plaque. The S11 strain showed 99.5% similarity with Lactobacillus fermentum, and was identified on the basis of biochemical characteristics and a 16S rDNA sequence. S11 strain and its culture supernatant significantly inhibited the formation of the insoluble glucan produced by Streptococcus mutans Ingbritt. S11 did not affect the multiplication of S. mutans Ingbritt, but the adherence of S. mutans Ingbritt onto cuvette walls was inhibited completely.     

Escape from the competence state in Streptococcus mutans is governed by the bacterial population density

Horizontal gene transfer through natural DNA transformation is an important evolutionary mechanism among bacteria. Transformation requires that the bacteria are physiologically competent to take and incorporate free DNA directly from the environment. Although natural genetic transformation is a remarkable feature of many naturally competent bacteria, the process is energetically expensive for the cells. Consequently, a tight control of the competence state is necessary. The objective of the present work was to help decipher the molecular mechanisms regulating the escape from the competence state in Streptococcus mutans, the principal etiological agent responsible for tooth decay in humans. Our results showed that the cessation of competence in S. mutans was abrupt, and did not involve the accumulation of a competence inhibitor nor the depletion of a competence activator in the extracellular environment. The competence state was repressed at high cell population density via concomitant repression of sigX gene encoding the master regulator of the competence regulon. Co‐culture experiments performed with oral and non‐oral bacteria showed that S. mutans assesses its own population density and also the microbial density of its surroundings to regulate its competence escape. Interestingly, neither the intra‐species and extra‐species quorum‐sensing systems nor the other 13 two‐component regulatory systems identified in S. mutans were involved in the cell‐density‐dependent escape of the competence state. Altogether, our results suggest a complex mechanism regulating the competence shut‐off involving cell‐density‐dependent repression of sigX through an as yet undefined system, and possibly SigX protein stability.     

PCR detection of Scardovia wiggsiae in combination with Streptococcus mutans for early childhood caries‐risk prediction

The microbial factor is an important determinant in caries risk assessment. This study aimed to use detection, by PCR, of Scardovia wiggsiae, in combination with Streptococcus mutans, for the accurate prediction of caries risk in children. Detection of Lactobacillus, as a caries‐specific species, was also performed. Dental plaque, as well as infected dentine when available, was collected from children who were caries‐free (n = 30) or diagnosed with early childhood caries (n = 30), and the prevalence and abundance of S. wiggsiae and S. mutans were estimated using quantitative PCR. Lactobacillus was amplified by Lactobacillus genus‐specific primers and then sequenced. Both S. wiggsiae and S. mutans were concurrently detected in 19 children diagnosed with early childhood caries, but in none of the caries‐free children. The positive predictive value was 1 in children with S. wiggsiae‐ and S. mutans‐positive test results, compared with 0.58 when only S. mutans was detected and 0.9 when only S. wiggsiae was detected. The abundance of S. wiggsiae and S. mutans in infected dentine was higher than that in dental plaque from children. Diverse Lactobacillus species were observed in dental plaque but none appeared to be caries‐specific. In conclusion, the detection of S. wiggsiae in combination with S. mutans improves the positive predictive value and the specificity of the test.     

Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model

There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar‐overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited.     

Antigen I/II mediates interactions between Streptococcus mutans and Candida albicans

Streptococcus mutans and Candida albicans are frequently co‐isolated from dental plaque of children with early childhood caries (ECC) and are only rarely found in children without ECC, suggesting that these species interact in a manner that contributes to the pathogenesis of ECC. Previous studies have demonstrated that glucans produced by S. mutans are crucial for promoting the formation of biofilm and cariogenicity with C. albicans; however, it is unclear how non‐glucan S. mutans biofilm factors contribute to increased biofilm formation in the presence of C. albicans. In this study we examined the role of S. mutans antigen I/II in two‐species biofilms with C. albicans, and determined that antigen I/II is important for the incorporation of C. albicans into the two‐species biofilm and is also required for increased acid production. The interaction is independent of the proteins Als1 and Als3, which are known streptococcal receptors of C. albicans. Moreover, antigen I/II is required for the colonization of both S. mutans and C. albicans during co‐infection of Drosophila melanogaster in vivo. Taken together, these results demonstrate that antigen I/II mediates the increase of C. albicans numbers and acid production in the two‐species biofilm, representing new activities associated with this known S. mutans adhesin.