Biological functions of glucan‐binding protein B of Streptococcus mutans

Introduction: Streptococcus mutans has been implicated as a major causative agent of dental caries in humans. Bacterial components associated with the adhesion phase of S. mutans include glucosyltransferases, protein antigen C and proteins that bind glucan. At least four glucan‐binding proteins (Gbp) have been identified; GbpA, GbpB, GbpC and GbpD. Methods: In our previous study, the contributions of GbpA and GbpC to the virulence of S. mutans were investigated; however, the biological function of GbpB and its role in the virulence of S. mutans remain to be elucidated. Using a GbpB‐deficient mutant strain (BD1), we demonstrated in the present study that GbpB has a role in the biology of S. mutans. Results: The growth rate of BD1 was lower than that of other strains, while it was also shown to be less susceptible to phagocytosis and to form longer chains than the parental strain MT8148. In addition, electron microscope observations of the cell surfaces of BD1 showed that the cell‐wall layers were obscure. Conclusion: These results suggest that GbpB may have an important role in cell‐wall construction and be involved in cell separation and cell maintenance.     

Streptococcus mutans binding to solid phase dextran mediated by the glucan‐binding protein C

Streptococcus mutans GbpC is a wall‐anchored surface protein which is involved in dextran‐dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N‐acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti‐GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(α‐1,6 glucan)‐dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.     

Comparison of glucan‐binding proteins in cariogenicity of Streptococcus mutans

Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. Bacterial components associated with the adhesion phase of S. mutans include cell‐associated and cell‐free glucosyltransferases (GTFs), as well as protein antigen c and proteins that bind glucan. At least four types of S. mutans glucan‐binding protein (Gbp) have been identified; GbpA, GbpB, GbpC and GbpD. In the present study, GbpA‐, GbpB‐ and GbpC‐deficient mutants (AD1, BD1 and CD1, respectively) were constructed, and their cariogenic properties were evaluated by comparing them to those of their parent strain MT8148. All of the Gbp mutants showed lower levels of dextran binding, while the sucrose‐dependent adhesion levels of AD1 and CD1 were lower than in the parental strain. The expression of each GTF was detected in the Gbp mutants, however, they had lower levels of cell‐free‐GTF activity than the parental strain. On the other hand, in acid tolerance assays, BD1 was the most sensitive among all of the tested strains. These results suggest that GbpA and GbpC in S. mutans have strong relationships with cariogenicity, while GbpB may have another biological function.     

Binding of glucan‐binding protein C to GTFD‐synthesized soluble glucan in sucrose‐dependent adhesion of Streptococcus mutans

Streptococcus mutans produces glucan‐binding proteins (Gbp proteins) which promote the adhesion of the organism to teeth. Three Gbp proteins, GbpA protein, GbpB protein, and GbpC protein have been identified; however, the mechanism of adhesion between glucans and bacterial cell surfaces is unknown. We used glucosyltransferase (GTF)‐ and/or Gbp‐deficient mutants to examine the role of GbpC protein in the sucrose‐dependent cellular adhesion of S. mutans to glass surfaces. The wild‐type strain MT8148 and a GbpA‐deficient mutant strain displayed increased sucrose‐dependent adhesion following the addition of rGTFD. However, a GbpC‐deficient mutant strain demonstrated no changes in the level of sucrose‐dependent adhesion in spite of the addition of rGTFD. Further, the binding of rGbpC protein to the glucan synthesized by rGTFD was significantly higher than that to the glucan synthesized by either rGTFB or rGTFC. These results suggest that GbpC protein may play an important role in sucrose‐dependent adhesion by binding to the soluble glucan synthesized by GTFD.     

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.     

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.     

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.     

Identification of a glucan‐binding protein C gene homologue in Streptococcus macacae

Background/aims: The past few decades have seen the isolation of certain glucosyltransferases and a number of proteins from mutans streptococci. Some of these proteins have been shown to possess glucan‐binding capabilities which confer an important virulence property on mutans streptococci for the role of these bacteria play in dental caries. Among these proteins is glucan‐binding protein C, which is encoded by the gbpC gene, and which we have identified as being involved in the dextran‐dependent aggregation of Streptococcus mutans. However, gbpC homologues have yet to be identified in other mutans streptococci. Methods: We carried out polymerase chain reaction amplification of Streptococcus macacae using primers that were designed based on conserved sequences of S. mutans gbpC and identified a gbpC gene homologue. The gene of that homologue was then characterized. Results: Nucleotide sequencing of the S. macacae gbpC homologue revealed a 1854 bp open reading frame encoding a protein with an N‐terminal signal peptide. The molecular mass of the processed protein was calculated to be 67 kDa. We also found an LPxTG motif, the consensus sequence for gram‐positive cocci cell wall‐anchored surface proteins, which was followed by a characteristic sequence at the carboxal terminal region of the putative protein. This suggests that the S. macacae GbpC homologue protein was tethered to the cell wall. Conclusion: Based on these results, together with the demonstrated glucan‐binding ability of the S. macacae GbpC homologue protein, we suggest that S. macacae cells are capable of binding dextran via the GbpC homologue protein, which is similar to the S. mutans GbpC protein. In addition, Southern hybridization analysis using the S. macacae gbpC homologue as a probe showed a distribution of gbpC homologues throughout the mutans streptococci.     

Influence of surface properties of resin‐based composites on in vitro Streptococcus mutans biofilm development

The aim of this in vitro study was to evaluate the influence of physicochemical surface properties of resin‐based composites on Streptococcus mutans biofilm formation. Specimens were prepared from each of four resin‐based composites by polymerization against Mylar strips. Half of the number of specimens received no further surface treatment, whereas the other half were subjected to a polishing treatment. Surface roughness (SR) and topography were assessed using profilometry and atomic force microscopy. Surface free‐energy (SFE) was determined, and the chemical surface composition was analysed by X‐ray photoelectron spectroscopy (XPS). S. mutans biofilms were formed on the surface of the resin‐based composite specimens for either 48 or 96 h using an artificial mouth system (AMS). Polishing caused a significant decrease in SFE, and XPS analysis indicated an increase of surface silicon and a decrease of surface carbon. Only for Grandio was a significant increase in SR identified after polishing, which was probably related to the higher concentration of filler particles on its surface. Significantly less S. mutans biofilm formation was observed on polished resin‐based composites than on unpolished resin‐based composites. These results indicate that the proportions of resin matrix and filler particles on the surface of resin‐based composites strongly influence S. mutans biofilm formation in vitro, suggesting that minimization of resin matrix exposure might be useful to reduce biofilm formation on the surface of resin‐based composites.     

Streptococcus mutans serotypes and collagen‐binding proteins Cnm/Cbm in children with caries analysed by PCR

Streptococcus mutans, a primary bacterium associated with dental caries, has four known clinical serotypes (c, e, fand k). Certain serotypes, the presence of multiple serotypes and strains with collagen‐binding proteins (CBP, Cnm and Cbm) have been linked with systemic disease. Evaluation of S mutans serotype distribution and caries association is needed in the United States. The purpose of this study was to evaluate the prevalence of S mutans serotypes from two cohorts of African‐American children in rural Alabama using three sample types (saliva, plaque and individual S mutans isolates) by PCR detection for association with caries. Detection of CBP was also performed by PCR. In total, 129 children were evaluated and overall prevalence of serotypes were: serotype c(98%), e(26%), f(7%) and k(52%). Serotype c was statistically associated with higher caries scores in older children (P < 0.001) and serotype k was statistically more likely in females (P = 0.004). Fourteen per cent of children had CBP. Thirteen S mutans isolates from five children tested positive for both CBP. This study is the first to report on the prevalence of S mutans serotypes in a US population using the PCR‐based approach. The frequency of serotype k in this study is the highest reported in any population, illustrating the need for further study to determine the prevalence of this clinically relevant serotype in the US. This is the first study to report S mutans isolates with both Cnm and Cbm in the same strain, and further analysis is needed to determine the clinical significance of these strains.     

Cnm is a major virulence factor of invasive Streptococcus mutans and part of a conserved three‐gene locus

Cnm, a collagen‐ and laminin‐binding protein present in a subset of Streptococcus mutans strains, mediates binding to extracellular matrices (ECM), intracellular invasion and virulence in the Galleria mellonella model. Antibodies raised against Cnm were used to confirm expression and the cell surface localization of Cnm in the highly invasive OMZ175 strain. Sequence analysis identified two additional genes (cnaB and cbpA) encoding putative surface proteins immediately upstream of cnm. Inactivation of cnaB and cbpA in OMZ175, individually or in combination, did not decrease the ability of this highly invasive and virulent strain to bind to different ECM proteins, invade human coronary artery endothelial cells (HCAEC), or kill G. mellonella. Similarly, expression of cnaB and cbpA in the cnm^? strain UA159 revealed that these genes did not enhance Cnm‐related phenotypes. However, integration of cnm in the chromosome of UA159 significantly increased its ability to bind to collagen and laminin, invade HCAEC, and kill G. mellonella. Moreover, the presence of antibodies against Cnm nearly abolished the ability of OMZ175 to bind to collagen and laminin and invade HCAEC, and significantly protected G. mellonella against OMZ175 infection. We concluded that neither CnaB nor CbpA is necessary for the expression of Cnm‐related traits. We also provided definitive evidence that Cnm is an important virulence factor and a suitable target for the development of novel preventive and therapeutic strategies to combat invasive S. mutans strains.     

Sublingual immunization with the phosphate‐binding‐protein (PstS) reduces oral colonization by Streptococcus mutans

Bacterial ATP‐binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate‐binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat‐labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A‐secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries.     

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.     

Inhibiting effects of Streptococcus salivarius on competence‐stimulating peptide‐dependent biofilm formation by Streptococcus mutans

Introduction: The effects of Streptococcus salivarius on the competence‐stimulating peptide (CSP)‐dependent biofilm formation by Streptococcus mutans were investigated. Methods: Biofilms were grown on 96‐well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm. Results: S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC‐3 and FSC‐3ΔglrA in separate dual‐species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP‐binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene‐dependent quorum sensing systems. Conclusion: It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell‐to‐cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.     

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.