Lactobacilli and human dental caries: more than mechanical retention.

Lactobacilli have been considered as major contributors to human dental caries for over a century. Recent in vitro model studies have shown that when compared to Streptococcus mutans, a keystone pathogen of human dental caries, the ability of lactobacilli to form biofilms is poor, although differences exist between the different major species. Further studies using molecular and bioinformatics approaches provide evidence that multiple mechanisms, including adhesin-receptor mediated physical contact with S. mutans, facilitate the adherence and establishment of lactobacilli on the tooth surface. There is also evidence that under conditions like continuous sugar consumption, weak acids and other antimicrobials such as bacteriocins from lactobacilli can become detrimental to the microbial community, especially those in the proximity. Details on the underlying mechanisms of how different Lactobacillus sp. establish and persist in the highly complex microbiota on the tooth surface await further investigation.

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.

The Impacts of Sortase A and the 4'-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis.

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on Streptococcus mutans EMV biogenesis of Sortase A (SrtA), which affects S. mutans EMV composition, and Sfp, a 4'-phosphopantetheinyl transferase that affects Bacillus subtilis EMV stability. ΔsrtA EMVs were notably larger than Δsfp and wild-type (WT) EMVs. EMV proteins identified from all three strains are known to be involved in cell wall biogenesis and cell architecture, bacterial adhesion, biofilm cell density and matrix development, and microbial competition. Notably, the AtlA autolysin was not processed to its mature active form in the ΔsrtA mutant. Proteomic and lipidomic analyses of all three strains revealed multiple dissimilarities between vesicular and corresponding cytoplasmic membranes (CMs). A higher proportion of EMV proteins are predicted substrates of the general secretion pathway (GSP). Accordingly, the GSP component SecA was identified as a prominent EMV-associated protein. In contrast, CMs contained more multi-pass transmembrane (TM) protein substrates of co-translational transport machineries than EMVs. EMVs from the WT, but not the mutant strains, were enriched in cardiolipin compared to CMs, and all EMVs were over-represented in polyketide flavonoids. EMVs and CMs were rich in long-chain saturated, monounsaturated, and polyunsaturated fatty acids, except for Δsfp EMVs that contained exclusively polyunsaturated fatty acids. Lipoproteins were less prevalent in EMVs of all three strains compared to their CMs. This study provides insight into biophysical characteristics of S. mutans EMVs and indicates discrete partitioning of protein and lipid components between EMVs and corresponding CMs of WT, ΔsrtA, and Δsfp strains.

Analysis of Fluoride Content in Alternative Milk Beverages.

Background: The dentist has a responsibility to provide nutritional counseling and fluoride consumption recommendations. The purpose of this study was to measure and compare the concentrations of fluoride in a large number of alternative milk beverages and bovine milk. Study design: Thirty-three milk alternatives, including 9 diverse types and 11 different brands, were analyzed using a fluoride ion-selective electrode (ISE) and an ISE meter. Fluoride concentrations were then compared among different types and between different brands. Results: Fluoride concentration ranged from 0.01 ppm (Malk® Pure Cashew Milk) to 0.80 ppm (Almond Breeze® Original Unsweetened Almond Milk) with a mean concentration of 0.32 ppm. When compared, bovine whole milk (0.03±0.00 ppm) was found to be significantly lower in fluoride than all samples analyzed except Malk Pure Cashew Milk, Soy Milk Vanilla, Rice Milk, and Pecan Milk. Major differences also existed between the same milk alternative types of different brands. Conclusion: The amount of fluoride varies among different types of milk alternatives and different brands. To ensure that the dental team can provide proper recommendations regarding fluoride use, manufacturers should consider placing fluoride concentrations on nutrition labels.

Analysis of cariogenic potential of alternative milk beverages by in vitro Streptococcus mutans biofilm model and ex vivo caries model.

OBJECTIVE: To evaluate the potential of various alternative milk beverages to support bacterial biofilm formation and acid production and cause unbalanced demineralization. DESIGN: in vitro assays were used to examine the ability of the beverages to support Streptococcus mutans' biofilm formation and acid production from sugar fermentation and the capacity of the beverages to buffer pH changes. Biofilm formation was done using 96-well plate model. Acid production was measured using L-Lactate assay kit, and the buffering capacity was assessed by pH titration. For ex vivo caries model, enamel and dentine slabs and S. mutans biofilms were exposed to selected alternative milk beverages three times a day, 30 min each, and by the end of the experiments, slab's demineralization was assessed by loss of surface microhardness. RESULTS: Of the alternative milk beverages tested in this study, Original Almond consistently supported the most S. mutans biofilms, followed by Chocolate Cashew Milk, while the least biofilms were measured with Unsweetened Flax Milk. The most acids and the lowest culture pH were measured with Toasted Coconut Almond Milk, while the least buffering capacity was measured with Unsweetened Coconut Milk. The results of ex vivo caries model showed that like Bovine Whole Milk, repeated exposure to Original Almond led to significant enamel and dentine slab demineralization, when compared to those exposed to saline as a control (P < 0.001). CONCLUSIONS: These results further provide support that popular alternative milk beverages, especially those with supplemental sugars, are potentially cariogenic.

Glycosyltransferase-Mediated Biofilm Matrix Dynamics and Virulence of Streptococcus mutans.

Streptococcus mutans is a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU_833, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu_833 decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU_833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion of gtfBC from the smu_833 mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat model of dental caries, demonstrating that SMU_833 is a new virulence factor. Taken together, we conclude that SMU_833 is required for optimal biofilm development and virulence of S. mutans by modulating extracellular matrix components. Our study of SMU_833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries.IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus mutans The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth's surface and causing demineralization and decay of teeth. As knowledge of our body's microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. mutans In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases.

Deficiency of MecA in Streptococcus mutans Causes Major Defects in Cell Envelope Biogenesis, Cell Division, and Biofilm Formation.

MecA is an adaptor protein that guides the ClpC/P-mediated proteolysis. A S. mutans MecA-deficient mutant was constructed by double-crossover allelic exchange and analyzed for the effects of such a deficiency on cell biology and biofilm formation. Unlike the wild-type, UA159, the mecA mutant, TW416, formed mucoid and smooth colonies, severely clumped in broth and had a reduced growth rate. Transmission electron microscopy analysis revealed that TW416 grows primarily in chains of giant "swollen" cells with multiple asymmetric septa, unlike the coccoid form of UA159. As compared to UA159, biofilm formation by TW416 was significantly reduced regardless of the carbohydrate sources used for growth (P < 0.001). Western blot analysis of TW416 whole cell lysates showed a reduced expression of the glucosyltransferase GtfC and GtfB, as well as the P1 and WapA adhesins providing an explanation for the defective biofilm formation of TW416. When analyzed by a colorimetric assay, the cell wall phosphate of the mutant murein sacculi was almost 20-fold lower than the parent strain (P < 0.001). Interestingly, however, when analyzed using immunoblotting of the murein sacculi preps with UA159 whole cell antiserum as a probe, TW416 was shown to possess significantly higher signal intensity as compared to the wild-type. There is also evidence that MecA in S. mutans is more than an adaptor protein, although how it modulates the bacterial pathophysiology, including cell envelope biogenesis, cell division, and biofilm formation awaits further investigation.

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.

Analysis of the Cariogenic Potential of Various Almond Milk Beverages using a Streptococcus mutans Biofilm Model in vitro.

To evaluate the cariogenic properties of almond milk beverages, 6 almond milks, along with soy and whole bovine milk, were analyzed for their abilities to support Streptococcus mutans biofilm formation and acid production, and their capacity to buffer changes in pH. Biofilm formation by S. mutans was analyzed using an in vitro 96-well plate model and measured by crystal violet staining. Acid production by S. mutans was evaluated by a colorimetric L-lactate assay and pH measurement of bacterial cultures. Buffering capacity was assessed by a pH titration assay. Soy milk supported the most biofilm growth, while the least was observed with unsweetened almond milk (both p < 0.001). Among almond milks, sucrose-sweetened milk led to the highest level of biofilm formation (p < 0.001), while the least was observed with unsweetened milk (p < 0.05). Sucrose-sweetened almond milk yielded the lowest pH (4.56 ± 0.66), followed by soy milk and bovine milk; the highest pH was with unsweetened almond milk (6.48 ± 0.5). When analyzed by pH titration, the unsweetened almond milk displayed the weakest buffering capacity while bovine milk showed the highest (p < 0.001). These results suggest that the almond milk beverages, except those that are sweetened with sucrose, possess limited cariogenic properties, while soy milk exhibits the most cariogenic potential. As milk alternatives become increasingly popular, dentists must counsel their patients that almond milks, especially sucrose-sweetened varieties, have cariogenic potential. For patients who are lactose-intolerant or suffer from milk allergy, almond milks may be a better alternative than soy-based products.

Synthesis, antibacterial activity, and biocompatibility of new antibacterial dental monomers.

PURPOSE: To synthesize a small library of antibacterial dental monomers based on quaternary ammonium salts and to test their antibacterial activity against cariogenic bacteria. METHODS: Five new antibacterial monomers were synthesized and characterized by NMR, IR and HRMS. RESULTS: Cytotoxicity assays using human gingival fibroblast cells showed that these new antibacterial monomers were biocompatible at concentrations of 10⁻5 M and displayed less cytotoxicity than BisGMA, a common dental monomer. When analyzed in vitro, all new monomers demonstrated strong inhibitory activity against biofilm formation by cariogenic Streptococcus mutans and Lactobacillus casei. Results indicated that antibacterial monomers containing a long alkyl (i.e. hexadecyl) chain are superior to their shorter-chain counterparts. The cross-linking monomers based on glycerol dimethacrylate also consistently outperformed their monomethacrylate analogs. Finally, the ammonium salts containing the dimethylbenzyl moiety were superior to the similar structures containing 1,4-diazabicyclo[2.2.2]octane (DABCO) in some cases. CLINICAL SIGNIFICANCE: All five new monomers were deemed biocompatible at concentrations of 10⁻5 M or less, and most had better biocompatibility than BisGMA. Dimethacrylate monomers 5 and 6 generally demonstrated high antibacterial activities, with the highest activity shown for the most lipophilic monomer 6, and these new antibacterial monomers have potential future application in dental composites and bonding agents.

Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG, encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation (P < 0.01). Double and triple mutants with deficiency in brpA and/or psr, genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically (P < 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope.IMPORTANCEStreptococcus mutans, a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans, indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans, but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics.

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c.

Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1- and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

Photo-cross-linked Antibacterial Zein Nanofibers Fabricated by Reactive Electrospinning and its Effects against Streptococcus mutans.

Native zein electrospun nanofibers have shown poor solvent resistance and low mechanical strength. Compared to other toxic cross-linkers, a safer method of stabilizing zein based fibers while retaining or with improved mechanical strength is needed to convert these materials for biomedical applications where culture media or body fluids may be present. We report here a method of fabricating non-toxic zein nanofibers using reactive electrospinning coupled with in situ photo-cross-linking. The cross-linked zein nanofibers exhibited significantly improved mechanical strength and sustained morphology against water and aqueous ethanol solution. This process doesn't require additional conventional cross-linking agents to form cross-linking network, which is advantageous for biomedical applications. Antimicrobial monomer with photo-reactive moiety was coupled with methacrylate zein nanofibers and showed strong inhibitory activity against cariogenic Streptococcus mutans. Cytotoxicity test with human gingival fibroblasts revealed high biocompatibility.

Streptococcus mutans Displays Altered Stress Responses While Enhancing Biofilm Formation by Lactobacillus casei in Mixed-Species Consortium.

Like Streptococcus mutans, lactobacilli are commonly isolated from carious sites, although their exact role in caries development remains unclear. This study used mixed-species models to analyze biofilm formation by major groups of oral lactobacilli, including L. casei, L. fermentum, L. rhamnosus, L. salivarius ssp. salivarius, and L. gasseri. The results showed that lactobacilli did not form good biofilms when grown alone, although differences existed between different species. When grown together with S. mutans, biofilm formation by L. gasseri and L. rhamnosus was increased by 2-log (P < 0.001), while biofilms by L. fermentum reduced by >1-log (P < 0.001). L. casei enhanced biofilm formation by ~2-log when grown with S. mutans wild-type, but no such effects were observed with S. mutans deficient of glucosyltransferase GtfB and adhesin P1. Both S. mutans and L. casei in dual-species enhanced resistance to acid killing with increases of survival rate by >1-log (P < 0.001), but drastically reduced the survival rates following exposure to hydrogen peroxide (P < 0.001), as compared to the respective mono-species cultures. When analyzed by RNA-seq, more than 134 genes were identified in S. mutans in dual-species with L. casei as either up- or down-regulated when compared to those grown alone. The up-regulated genes include those for superoxide dismutase, NADH oxidase, and members of the mutanobactin biosynthesis cluster. Among the down-regulated genes were those for GtfB and alternative sigma factor SigX. These results further suggest that interactions between S. mutans and oral lactobacilli are species-specific and may have significant impact on cariogenic potential of the community.

Fabrication and Characterization of a Glucose-sensitive Antibacterial Chitosan-Polyethylene Oxide Hydrogel.

A novel glucose-sensitive chitosan-polyethylene oxide (CS/PEO =1:0.5~1:2.5) hydrogel with controlled release of metronidazole (MNZ) was obtained by chemical cross-linking and immobilization of glucose oxidase (GOx). The hydrogel was characterized by Fourier-transformed infrared spectroscopy (FTIR), compressive mechanical test, rheological analysis, cytotoxicity test, and antibacterial test against Porphyromonas gingivalis. The study found that the CS-PEO composite hydrogel possessed significantly better mechanical properties and biocompatibility than a single-component hydrogel. This might result from the physical cross-linking and formation of semi-interpenetrating network (semi-IPN). In addition, this novel hydrogel has self-regulate ability to release MNZ in response to the environmental glucose stimulus. Specifically, it released more drugs at higher glucose concentration, thus can lead to a greater ability to inhibit Porphyromonas gingivalis. This study has demonstrated the glucose-sensitive antibacterial hydrogel has a great potential as a new therapeutic material for treatment or prevention of periodontitis in diabetic patients.

PBP1a-deficiency causes major defects in cell division, growth and biofilm formation by Streptococcus mutans.

Streptococcus mutans, a key etiological agent of human dental caries, lives almost exclusively on the tooth surface in plaque biofilms and is known for its ability to survive and respond to various environmental insults, including low pH, and antimicrobial agents from other microbes and oral care products. In this study, a penicillin-binding protein (PBP1a)-deficient mutant, strain JB467, was generated by allelic replacement mutagenesis and analyzed for the effects of such a deficiency on S. mutans' stress tolerance response and biofilm formation. Our results so far have shown that PBP1a-deficiency in S. mutans affects growth of the deficient mutant, especially at acidic and alkaline pHs. As compared to the wild-type, UA159, the PBP1a-deficient mutant, JB467, had a reduced growth rate at pH 6.2 and did not grow at all at pH 8.2. Unlike the wild-type, the inclusion of paraquat in growth medium, especially at 2 mM or above, significantly reduced the growth rate of the mutant. Acid killing assays showed that the mutant was 15-fold more sensitive to pH 2.8 than the wild-type after 30 minutes. In a hydrogen peroxide killing assay, the mutant was 16-fold more susceptible to hydrogen peroxide (0.2%, w/v) after 90 minutes than the wild-type. Relative to the wild-type, the mutant also had an aberrant autolysis rate, indicative of compromises in cell envelope integrity. As analyzed using on 96-well plate model and spectrophotometry, biofilm formation by the mutant was decreased significantly, as compared to the wild-type. Consistently, Field Emission-SEM analysis also showed that the PBP1a-deficient mutant had limited capacity to form biofilms. TEM analysis showed that PBP1a mutant existed primarily in long rod-like cells and cells with multiple septa, as compared to the coccal wild-type. The results presented here highlight the importance of pbp1a in cell morphology, stress tolerance, and biofilm formation in S. mutans.

Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery.

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans.

Streptococcus mutans, the primary aetiological agent of dental caries, possesses an YjeE-like protein that is encoded by locus SMU.409, herein designated brpB. In this study, a BrpB-deficient mutant, JB409, and a double mutant deficient of BrpB and BrpA (a paralogue of the LytR-CpsA-Psr family of cell wall-associated proteins), JB819, were constructed and characterized using function assays and microscopy analysis. Both JB409 and JB819 displayed extended lag phases and drastically slowed growth rates during growth in brain heart infusion medium as compared to the wild-type, UA159. Relative to UA159, JB409 and JB819 were more than 60- and 10-fold more susceptible to acid killing at pH 2.8, and more than 1 and 2 logs more susceptible to hydrogen peroxide, respectively. Complementation of the deficient mutants with a wild-type copy of the respective gene(s) partly restored the acid and oxidative stress responses to a level similar to the wild-type. As compared to UA159, biofilm formation by JB409 and JB819 was drastically reduced (P<0.001), especially during growth in medium containing sucrose. Under a scanning electron microscope, JB409 had significantly more giant cells with an elongated, rod-like morphology, and JB819 formed marble-like super cells with apparent defects in cell division. As revealed by transmission electron microscopy analysis, BrpB deficiency in both JB409 and JB819 resulted in the development of low electron density patches and formation of a loose nucleoid structure. Taken together, these results suggest that BrpB likely functions together with BrpA in regulating cell envelope biogenesis/homeostasis in Strep. mutans. Further studies are under way to elucidate the mechanism that underlies the BrpA- and BrpB-mediated regulation.

Psr is involved in regulation of glucan production, and double deficiency of BrpA and Psr is lethal in Streptococcus mutans.

Streptococcus mutans, the primary causative agent of dental caries, contains two paralogues of the LytR-CpsA-Psr family proteins encoded by brpA and psr, respectively. Previous studies have shown that BrpA plays an important role in cell envelope biogenesis/homeostasis and affects stress responses and biofilm formation by Strep. mutans, traits critical to cariogenicity of this bacterium. In this study, a Psr-deficient mutant, TW251, was constructed. Characterization of TW251 showed that deficiency of Psr did not have any major impact on growth rate. However, when subjected to acid killing at pH 2.8, the survival rate of TW251 was decreased dramatically compared with the parent strain UA159. In addition, TW251 also displayed major defects in biofilm formation, especially during growth with sucrose. When compared to UA159, the biofilms of TW251 were mainly planar and devoid of extracellular glucans. Real-time-PCR and Western blot analyses revealed that deficiency of Psr significantly decreased the expression of glucosyltransferase C, a protein known to play a major role in biofilm formation by Strep. mutans. Transmission electron microscopy analysis showed that deficiency of BrpA caused alterations in cell envelope and cell division, and the most significant defects were observed in TW314, a Psr-deficient and BrpA-down mutant. No such effects were observed with Psr mutant TW251 under similar conditions. These results suggest that while there are similarities in functions between BrpA and Psr, distinctive differences also exist between these two paralogues. Like Bacillus subtilis but different from Staphylococcus aureus, a functional BrpA or Psr is required for viability in Strep. mutans.

The redox-sensing regulator Rex modulates central carbon metabolism, stress tolerance response and biofilm formation by Streptococcus mutans.

The Rex repressor has been implicated in regulation of central carbon and energy metabolism in gram-positive bacteria. We have previously shown that Streptococcus mutans, the primary causative agent of dental caries, alters its transcriptome upon Rex-deficiency and renders S. mutans to have increased susceptibility to oxidative stress, aberrations in glucan production, and poor biofilm formation. In this study, we showed that rex in S. mutans is co-transcribed as an operon with downstream guaA, encoding a putative glutamine amidotransferase. Electrophoretic mobility shift assays showed that recombinant Rex bound promoters of target genes avidly and specifically, including those down-regulated in response to Rex-deficiency, and that the ability of recombinant Rex to bind to selected promoters was modulated by NADH and NAD(+). Results suggest that Rex in S. mutans can function as an activator in response to intracellular NADH/NAD(+) level, although the exact binding site for activator Rex remains unclear. Consistent with a role in oxidative stress tolerance, hydrogen peroxide challenge assays showed that the Rex-deficient mutant, TW239, and the Rex/GuaA double mutant, JB314, were more susceptible to hydrogen peroxide killing than the wildtype, UA159. Relative to UA159, JB314 displayed major defects in biofilm formation, with a decrease of more than 50-fold in biomass after 48-hours. Collectively, these results further suggest that Rex in S. mutans regulates fermentation pathways, oxidative stress tolerance, and biofilm formation in response to intracellular NADH/NAD(+) level. Current effort is being directed to further investigation of the role of GuaA in S. mutans cellular physiology.