Silver Diammine Fluoride Usage in General Dentistry Offices in Louisiana.

Purpose: To evaluate the current knowledge and usage of silver diammine fluoride (SDF) by general dentists in Louisiana and to identify primary barriers to the imple- mentation of SDF. Methods: A 16-item survey was emailed to 1719 Louisiana Dental Association members to identify factors influencing general dentists' usage of SDF. Results: Eighty-two surveys were completed with a response rate of 4.8 percent, with 69 identified as general dentists. Over half of the respondents were male (53.6%) and their practice experience ranged from less than one year to 48 years. The majority were solo owners (43.5%) while 7.3 percent had jobs in the corporate setting. Most agreed/strongly agreed that their knowledge of SDF was from either dental journals or online resources, while fewer stated they were taught about SDF (25%) or used SDF (8%) in dental school. The majority knew the advantages and off-label usage of SDF. However, only 40 percent recognized that SDF was officially approved for tooth hypersensitivity only. The most reported perceived barrier to SDF implementation was not learning about SDF in dental school (36%). Conclusion: There is a lack of understanding of SDF usage among Louisiana general dentists. The main reason for not incorporating SDF into their practice is the lack of training in their dental education.

Impacts of a DUF2207 Family Protein on Streptococcus mutans Stress Tolerance Responses and Biofilm Formation.

Locus SMU.243 in Streptococcus mutans was annotated as a member of the DUF2207 family proteins highly conserved in all bacteria but with unknown function. To investigate its role in S. mutans physiology, a SMU.243-deficient mutant was constructed using allelic exchange mutagenesis, and the impacts of SMU.243 deletion on bacterial growth, stress tolerance response, and biofilm formation were analyzed. Compared to the wild-type UA159, S. mutans lacking SMU.243 displayed a reduced growth rate and a reduced overnight culture density (p < 0.01) when grown at low pH and in the presence of methyl viologen. Relative to the parent strain, the deficient mutant also had a reduced survival rate following incubation in a buffer of pH 2.8 (p < 0.01) and in a buffer containing hydrogen peroxide at 58 mM after 60 min (p < 0.001) and had a reduced capacity in biofilm formation especially in the presence of sucrose (p < 0.01). To study any ensuing functional/phenotypical links between SMU.243 and uppP, which is located immediately downstream of SMU.243 and encodes an undecaprenyl pyrophosphate phosphatase involved in recycling of carrier lipid undecaprenyl phosphate, a uppP deficient mutant was generated using allelic exchange mutagenesis. Unlike the SMU.243 mutant, deletion of uppP affected cell envelope biogenesis and caused major increases in susceptibility to bacitracin. In addition, two variant morphological mutants, one forming rough colonies and the other forming mucoid, smooth colonies, also emerged following the deletion of uppP. The results suggest that the SMU.243-encoded protein of the DUF2207 family in S. mutans plays an important role in stress tolerance response and biofilm formation, but unlike the downstream uppP, does not seem to be involved in cell envelope biogenesis, although the exact roles in S. mutans' physiology awaits further investigation.

Direct Comparison Between Intraoral Scanning and Alginate Impressions for Pediatric Patients: An In Vitro Study.

Purpose: To compare the accuracy and efficiency of both alginate and digital impression techniques in a method representative of a clinical setting. By evaluating the difference in fabrication time and accuracy for these techniques, it can be determined if using digital scanning is a viable alternative to alginate impressions to fabricate pediatric dental appliances.
Methods: Both the alginate and digital impressions were taken from the same pediatric typodont study model. The alginate impressions were handled and poured up, per product instructions, and digital impressions were 3D printed. A digital caliper was used to measure different landmarks on both the stone models and 3D printed models, and these measurements were compared to the pediatric typodont to determine accuracy. All steps in the process, including obtaining the impression, fabrication of the model and total time, were recorded to determine the efficiency of both methods.
Results: The alginate models had a significantly higher intercanine distance than the control. Measurements of anterior-posterior (AP) left, AP right, left central, and right central were larger than the control but did not show a significant difference in measurement. The alginate group had a significantly shorter impression time. Model fabrication time between the two groups was similar.
Conclusion: Alginate impressions require less chairside time and the digital technique was shown to be accurate in all measurements. Digital scanning may be a good alternative to alginate impressions for pediatric patients.

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.

Streptococcus mutans Lacking sufCDSUB Is Viable, but Displays Major Defects in Growth, Stress Tolerance Responses and Biofilm Formation.

Streptococcus mutans appears to possess a sole iron-sulfur (Fe-S) cluster biosynthesis system encoded by the sufCDSUB cluster. This study was designed to examine the role of sufCDSUB in S. mutans physiology. Allelic exchange mutants deficient of the whole sufCDSUB cluster and in individual genes were constructed. Compared to the wild-type, UA159, the sufCDSUB-deficient mutant, Δsuf::kan r , had a significantly reduced growth rate, especially in medium with the absence of isoleucine, leucine or glutamate/glutamine, amino acids that require Fe-S clusters for biosynthesis and when grown with medium adjusted to pH 6.0 and under oxidative and nitrosative stress conditions. Relative to UA159, Δsuf::kan r had major defects in stress tolerance responses with reduced survival rate of > 2-logs following incubation at low pH environment or after hydrogen peroxide challenge. When compared to UA159, Δsuf::kan r tended to form aggregates in broth medium and accumulated significantly less biofilm. As shown by luciferase reporter fusion assays, the expression of sufCDSUB was elevated by > 5.4-fold when the reporter strain was transferred from iron sufficient medium to iron-limiting medium. Oxidative stress induced by methyl viologen increased sufCDSUB expression by > 2-fold, and incubation in a low pH environment led to reduction of sufCDSUB expression by > 7-fold. These results suggest that lacking of SufCDSUB in S. mutans causes major defects in various cellular processes of the deficient mutant, including growth, stress tolerance responses and biofilm formation. In addition, the viability of the deficient mutant also suggests that SUF, the sole Fe-S cluster machinery identified is non-essential in S. mutans, which is not known in any other bacterium lacking the NIF and/or ISC system. However, how the bacterium compensates the Fe-S deficiency and if any novel Fe-S assembly systems exist in this bacterium await further investigation.

Complete Genome Sequence of Streptococcus mutans 27-3, an Active Extracellular Membrane Vesicle Producer.

Here, we report the complete genome sequence of Streptococcus mutans 27-3. Isolated from a caries-active patient, 27-3 produces significantly more extracellular membrane vesicles than the commonly used laboratory strain UA159. This study provides useful information for comparative genomic analysis and better understanding of regulation of vesiculogenesis in this bacterium.

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.

Azalomycin F5a Eradicates Staphylococcus aureus Biofilm by Rapidly Penetrating and Subsequently Inducing Cell Lysis.

Antimicrobial resistance has emerged as a serious threat to public health. Bacterial biofilm, as a natural lifestyle, is a major contributor to resistance to antimicrobials. Azalomycin F5a, a natural guanidine-containing polyhydroxy macrolide, has remarkable activities against Gram-positive bacteria, including Staphylococcus aureus, a major causative agent of hospital-acquired infections. To further evaluate its potential to be developed as a new antimicrobial agent, its influence on S. aureus biofilm formation was evaluated using the crystal violet method, and then its eradication effect against mature biofilms was determined by confocal laser scanning microscopy, the drop plate method, and regrowth experiments. The results showed that azalomycin F5a could significantly inhibit S. aureus biofilm formation, and such effects were concentration dependent. In addition, it can also eradicate S. aureus mature biofilms with the minimum biofilm eradication concentration of 32.0 µg/mL. As extracellular deoxyribonucleic acid (eDNA) plays important roles in the structural integrity of bacterial biofilm, its influence on the eDNA release in S. aureus biofilm was further analyzed using gel electrophoresis. Combined with our previous works, these results indicate that azalomycin F5a could rapidly penetrate biofilm and causes damages to the cell membrane, leading to an increase in DNase release and eventually eradicating S. aureus biofilm.

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.

Formulation and characterization of antibacterial orthodontic adhesive.

OBJECTIVE: The objective of this study was to formulate experimental orthodontic bracket adhesives and test their mechanical properties, fluoride release and antibacterial activity. METHODS: Four experimental antibacterial orthodontic bracket adhesives were prepared with different compositions of synthesized antibacterial monomers replacing total 5% of dental monomers in the control Transbond XT (3M): 5%C11, 3.5%C11+1.5%C2, 5%C16, and 3.5%C16+1.5%C2. Transbond XT alone was used as control. These groups were used to bond premolar brackets to extracted premolars. Shear bond strength (SBS) was tested using an Instron machine. For antibacterial test, disk specimens (10mm diameter, 1mm thick, n=4) were fabricated and incubated with cultures of cariogenic Streptococcus mutans for 48h, and following gentle sonication, S. mutans biofilms in colony-forming-units (CFU) on the disks were enumerated by plating on agar medium. The data were analyzed using ANOVA and Tukey test (α=0.05). RESULTS: All experimental groups had similar shear bond strength (no significant difference) to the control. All experimental groups showed significant inhibitory effect against S. mutans biofilm formation, when compared to the control, but there was no significant difference between experimental groups. CONCLUSION: Antibacterial orthodontic adhesive can be fabricated to have similar mechanical properties but better caries-inhibitory effect than current adhesive.

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.