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.     

Genes responsible for dextran‐dependent aggregation of Streptococcus sobrinus strain 6715

Introduction: Streptococcus sobrinus exhibits more significant dextran‐dependent aggregation mediated by glucan‐binding proteins than Streptococcus mutans. We have identified four glucan‐binding protein C gene (gbpC) homologues designated as gbpC1, gbpC2, dblA and dblB in S. sobrinus in contrast to the single gene gbpC in S. mutans. We attempted to determine which gene is most responsible for the dextran‐dependent aggregation of S. sobrinus. Methods: We introduced mutation with a chemical mutagen, 1‐methyl‐3‐nitro‐1‐nitrosoguanidine, into S. sobrinus strain 6715 and analysed the four gbpC homologous gene sequences in the parental strain 6715 and an obtained aggregation‐negative mutant NUM‐Ssg99. We also examined the localization of proteins encoded by these genes in the mutant NUM‐Ssg99. Results: The nucleotide sequences of the gbpC1, gbpC2 and dblA genes in NUM‐Ssg99 were 100% identical to the homologous genes in parental strain 6715. In contrast, a truncated mutation was detected in the dblB gene and the mutant protein devoid of the LPXTG motif was confirmed by Western blot analysis to be released into the extracellular milieu. Conclusion: We conclude that the dblB gene among the four GbpC homologous protein genes is most responsible for aggregation in strain 6715.     

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.     

One of two gbpC gene homologues is involved in dextran‐dependent aggregation of Streptococcus sobrinus

Introduction: Streptococcus sobrinus exhibits marked dextran‐dependent aggregation mediated by glucan‐binding proteins (GBPs). In contrast to Streptococcus mutans, in which the gbpC gene responsible for dextran‐dependent aggregation of this organism has been characterized, genes encoding the S. sobrinus GBPs have not yet been identified. Methods: Recently, we identified the gbpC gene homologue from Streptococcus macacae using polymerase chain reaction primers based on the conserved regions of the gbpC sequence exhibiting intraspecies variations. This method was applied to amplify a S. sobrinus homologue. Results: Unexpectedly, two gbpC gene homologues were identified in S. sobrinus strain 100‐4. One homologue, named gbpC, was more similar to the S. mutans gbpC gene than the other and was approximately half the molecular size of its homologue with similar regions interrupted by several non‐similar stretches. However, the dextran‐binding activity of the protein expressed from gbpC in Escherichia coli was not detected in contrast to the other homologue, a protein designated as Dbl, expressing this activity. The gbpC gene was shown to be intact on the chromosome of strain OMZ176, which does not exhibit dextran‐dependent aggregation, while the dbl gene of this strain contained a single adenine nucleotide insertion at approximately one‐third the distance from the 5′‐end. The insertion mutation in the dbl gene resulted in translation of a premature protein missing its LPXTG sequence signature sequence of the wall‐anchored proteins. Conclusion: These results suggest that the dbl gene is very likely responsible for S. sobrinus dextran‐dependent aggregation.     

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.     

Multiple factors are involved in regulation of extracellular membrane vesicle biogenesis in Streptococcus mutans

Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4′‐phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74‐fold (p < .05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di‐adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p < .05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a >1.5‐fold (p < .05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.     

Streptococcus mutans SpaP binds to RadD of Fusobacterium nucleatum ssp. polymorphum

Adhesin‐mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species‐specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non‐mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram‐positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD‐SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species‐ and subspecies‐dependent adhesin interactions.     

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.     

Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans

Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram‐positive bacteria, including Bacillus and Lactococcus, encode functions involved in protein post‐export. In this study, we constructed a PrsA‐deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/membrane protein profile as well as cell‐surface‐related phenotypes, including auto‐aggregation, increased surface hydrophobicity and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface‐display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to those identified in Bacillus, and so is likely to be involved in protein post‐export.     

Deletion of csn2 gene affects acid tolerance and exopolysaccharide synthesis in Streptococcus mutans

Csn2 is an important protein of the CRISPR‐Cas system. The physiological function of this protein and its regulatory role in Streptococcus mutans, as the primary causative agent of human dental caries, is still unclear. In this study, we investigated whether csn2 deletion would affect S. mutans physiology and virulence gene expression. We used microscopic imaging, acid killing assays, pH drop, biofilm formation, and exopolysaccharide (EPS) production tests to determine whether csn2 deletion influenced S. mutans colony morphology, acid tolerance/production, and glucan formation abilities. Comparisons were made between quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) data from the UA159 and csn2 deletion strain to determine the impact of csn2 knockout on S. mutans gene expression. The results showed that deletion of S. mutans csn2 changed its colony morphotype and made it more sensitive to acid. The expression levels of aciduricity genes, including leuA, leuB, leuC, and leuD, were significantly down‐regulated. Acid adaptation restored the aciduricity of csn2 mutant and enhanced the ability to synthesize EPS. The expression levels of EPS synthesis‐related genes, including gtfC and gtfD, were significantly up‐regulated after acid adaptation. In summary, deletion of S. mutans csn2 exerted multiple effects on the virulence traits of this pathogen, including acid tolerance and EPS formation, and that these alterations could partially be attributed to changes in gene expression upon loss of csn2. Understanding the function of csn2 in S. mutans might lead to novel strategies to prevent or treat imbalances in oral microbiota that may favor diseases.     

gbpC and pac gene mutations detected in Streptococcus mutans strain GS‐5

The Streptococcus mutans gbpC gene encoding cell wall‐anchoring glucan‐binding protein C is involved in the dextran(alpha‐1,6 glucan)‐dependent aggregation (ddag) of this organism. Unlike cells of other strains of S. mutans, strain GS‐5 cells did not exhibit dextran(alpha‐1,6 glucan)‐dependent aggregation under any conditions. We therefore hypothesized that the gbpC gene may be mutated in strain GS‐5. Sequencing analysis of the 1752‐nucleotide GS‐5 gbpC gene revealed a point mutation that switched codon 65 to a TAA termination codon. Strain GS‐5 was previously reported also to have a mutation in the pac gene encoding the cell wall‐anchored major protein antigen. The laboratory‐maintained strain GS‐5 is regarded as having lower cariogenicity than the original isolate. The decreased cariogenicity developed during the laboratory culture of strain GS‐5 may have been caused by mutations in an environment lacking appropriate selective pressures.     

Streptococcus mutans copper chaperone,CopZ, is critical for biofilm formation and competitiveness

The oral cavity is a dynamic environment characterized by hundreds of bacterial species, saliva, and an influx of nutrients and metal ions such as copper. Although there is a physiologic level of copper in the saliva, the oral cavity is often challenged with an influx of copper ions. At high concentrations copper is toxic and must therefore be strictly regulated by pathogens for them to persist and cause disease. The cariogenic pathogen Streptococcus mutans manages excess copper using the copYAZ operon that encodes a negative DNA‐binding repressor (CopY), the P1‐ATPase copper exporter (CopA), and the copper chaperone (CopZ). These hypothetical roles of the copYAZ operon in regulation and copper transport to receptors led us to investigate their contribution to S. mutans virulence. Mutants defective in the copper chaperone CopZ, but not CopY or CopA, were impaired in biofilm formation and competitiveness against commensal streptococci. Characterization of the CopZ mutant biofilm revealed a decreased secretion of glucosyltransferases and reduced expression of mutacin genes. These data suggest that the function of copZ on biofilm and competitiveness is independent of copper resistance and CopZ is a global regulator for biofilm and other virulence factors. Further characterization of CopZ may lead to the identification of new biofilm pathways.     

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.     

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.     

Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans

Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining this balance. Using the six‐species Zürich “supragingival” biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony‐forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries‐favoring dysbiotic state.     

Extracellular matrix influence in Streptococcus mutans gene expression in a cariogenic biofilm

Caries etiology is biofilm–diet‐dependent. Biofilms are highly dynamic and structured microbial communities enmeshed in a three‐dimensional extracellular matrix. The study evaluated the expression dynamics of Streptococcus mutans genes associated with exopolysaccharides (EPS) (gtfBCD, gbpB, dexA), lipoteichoic acids (LTA) (dltABCD, SMU_775c) and extracellular DNA (eDNA) (lytST, lrgAB, ccpA) during matrix development within a mixed‐species biofilm of S. mutans, Actinomyces naeslundii and Streptococcus gordonii. Mixed‐species biofilms using S. mutans strains UA159 or ΔgtfB formed on saliva‐coated hydroxyapatite discs were submitted to a nutritional challenge (providing an abundance of sucrose and starch). Biofilms were removed at eight developmental stages for gene expression analysis by quantitative polymerase chain reaction. The pH of spent culture media remained acidic throughout the experimental periods, being lower after sucrose and starch exposure. All genes were expressed at all biofilm developmental phases. EPS‐ and LTA‐associated genes had a similar expression profile for both biofilms, presenting lower levels of expression at 67, 91 and 115 hours and a peak of expression at 55 hours, but having distinct expression magnitudes, with lower values for ΔgtfB (eg, fold‐difference of ~382 for gtfC and ~16 for dltB at 43 hours). The eDNA‐associated genes presented different dynamics of expression between both strains. In UA159 biofilms lrgA and lrgB genes were highly expressed at 29 hours (which were ~13 and ~5.4 times vs ΔgtfB, respectively), whereas in ΔgtfB biofilms an inverse relationship between lytS and lrgA and lrgB expression was detected. Therefore, the deletion of gtfB influences dynamics and magnitude of expression of genes associated with matrix main components.     

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans

We report the whole genome sequence of the serotype e Cbm⁺ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra‐oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall‐anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram‐positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.