Revisiting gas-chromatography/mass-spectrometry molar response factors for quantitative analysis (FID or TIC) of glycosidic linkages in polysaccharides produced by oral bacterial biofilms.

Methylation analysis was performed on methylated alditol acetate standards and Streptococcus mutans extracellular polymeric substances (EPS) produced from wild-type and Gtf knockout strains (∆GtfB, ∆GtfB, and ∆GtfD). The methylated alditol acetate standards were representative of glycosidic linkages found in S. mutans EPS and were used to calibrate the GC-MS system for an FID detector and MS (TIC) and produce molar response factor, a necessary step in quantitative analysis. FID response factors were consistent with literature values (Sweet et al., 1975) and found to be the superior option for quantitative results, although the TIC response factors now give researchers without access to an FID detector a needed option for molar response factor correction. The GC-MS analysis is then used to deliver the ratio of the linkage types within a biofilm.

Plasmalogen, a glycerophospholipid crucial for Streptococcus mutans acid tolerance and colonization.

Plasmalogen is a specific glycerophospholipid present in both animal and bacterial organisms. It plays a crucial function in eukaryotic cellular processes and is closely related to several human diseases, including neurological disorders and cancers. Nonetheless, the precise biological role of plasmalogen in bacteria is not well understood. In this study, we identified SMU_438c as the enzyme responsible for plasmalogen production in Streptococcus mutans under anaerobic conditions. The heterologous expression of SMU_438c in a plasmalogen-negative strain, Streptococcus sanguinis, resulted in the production of plasmalogen, indicating that this enzyme is sufficient for plasmalogen production. Additionally, the plasmalogen-deficient S. mutans exhibited significantly lower acid tolerance and diminished its colonization in Drosophila flies compared to the wild-type strain and complemented strain. In summary, our data suggest that plasmalogen plays a vital role in bacterial stress tolerance and in vivo colonization. IMPORTANCE: This study sheds light on the biological role of plasmalogen, a specific glycerophospholipid, in bacteria, particularly in Streptococcus mutans. Plasmalogens are known for their significant roles in eukaryotic cells and have been linked to human diseases like neurological disorders and cancers. The enzyme SMU_438c, identified as essential for plasmalogen production under anaerobic conditions, was crucial for acid tolerance and in vivo colonization in Drosophila by S. mutans, underscoring its importance in bacterial stress response and colonization. These findings bridge the knowledge gap in bacterial physiology, highlighting plasmalogen's role in microbial survival and offering potential insights into microbial pathogenesis and host-microbe interactions.

Oral bacterium Streptococcus mutans promotes tumor metastasis through thrombosis formation.

Thrombosis is a well-known cardiovascular disease (CVD) complication that has caused death in many patients with cancer. Oral bacteria have been reported to contribute to systemic diseases, including CVDs, and tumor metastasis. However, whether oral bacteria-induced thrombosis induces tumor metastasis remains poorly understood. In this study, the cariogenic oral bacterium Streptococcus mutans was used to examine thrombosis in vitro and in vivo. Investigation of tumor metastasis to the lungs was undertaken by intravenous S. mutans implantation using a murine breast cancer metastasis model. The results indicated that platelet activation, aggregation, and coagulation were significantly altered in S. mutans-stimulated endothelial cells (ECs), with elevated neutrophil migration, thereby inducing thrombosis formation. Streptococcus mutans stimulation significantly enhances platelet and tumor cell adhesion to the inflamed ECs. Furthermore, S. mutans-induced pulmonary thrombosis promotes breast cancer cell metastasis to the lungs in vivo, which can be reduced by using aspirin, an antiplatelet drug. Our findings indicate that oral bacteria promote tumor metastasis through thrombosis formation. Oral health management is important to prevent CVDs, tumor metastasis, and their associated death.

Transcriptome Analysis of Streptococcus mutans Quorum Sensing-Mediated Persisters Reveals an Enrichment in Genes Related to Stress Defense Mechanisms.

Persisters are a small fraction of growth-arrested phenotypic variants that can survive lethal concentrations of antibiotics but are able to resume growth once antibiotics are stopped. Their formation can be a stochastic process or one triggered by environmental cues. In the human pathogen Streptococcus mutans, the canonical peptide-based quorum-sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work has shown that the CSP-signaling peptide is a stress-signaling alarmone that promotes the formation of stress-induced persisters. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions and isolated the antibiotic persisters by treating the cultures with ofloxacin. A transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary-phase cells and the persisters. RNA sequencing revealed that triggered persistence was associated with the upregulation of genes related to several stress defense mechanisms, notably, multidrug efflux pumps, the arginine deaminase pathway, and the Opu/Opc system. In addition, we showed that inactivation of the VicK kinase of the YycFG essential two-component regulatory system abolished the formation of triggered persisters via the CSP pheromone. These data contribute to the understanding of the triggered persistence phenotype and may suggest new therapeutic strategies for treating persistent streptococcal infections.

Green Tea-Derived Catechins Suppress the Acid Productions of Streptococcus mutans and Enhance the Efficiency of Fluoride.

Green tea-derived catechins, which can be divided into galloylated (epicatechin gallate: ECG, epigallocatechin gallate: EGCG) and non-galloylated (catechin: C, epicatechin: EC, epigallocatechin: EGC) catechins, are considered to be the main contributors to the caries control potential of green tea. In this study, we intended to compare the antimicrobial effects of these representative green tea-derived catechins and their combined effects with fluoride on the acid production and aggregation of Streptococcus mutans. The effects of different catechins on the growth, aggregation and acid production of S. mutans, and the combined effect of catechins and potassium fluoride (2 mm at pH 7.0, 0.3 mm at pH 5.5) on S. mutans acid production were measured by anaerobic culture, turbidity changes due to aggregation, and pH-stat methods. Molecular docking simulations were also performed to investigate the interactions between catechins and membrane-embedded enzyme II complex (EIIC), a component of the phosphoenolpyruvate-dependent phosphotransferase system (sugar uptake-related enzyme). ECG or EGCG at 1 mg/mL significantly inhibited the growth of S. mutans, induced bacterial aggregation, and decreased glucose-induced acid production (p < 0.05). All catechins were able to bind to EIIC in silico, in the following order of affinity: EGCG, ECG, EGC, EC, and C. Furthermore, they enhanced the inhibitory effects of fluoride at pH 5.5 and significantly inhibited S. mutans acid production by 47.5-86.6% (p < 0.05). These results suggest that both galloylated and non-galloylated catechins exhibit antimicrobial activity, although the former type demonstrates stronger activity, and that the caries control effects of green tea may be due to the combined effects of multiple components, such as catechins and fluoride. The detailed mechanisms underlying these phenomena and the in vivo effect need to be explored further.

Streptococcus mutans sigX-inducing peptide inhibits the virulence of Candida albicans and oral candidiasis through the Ras1-cAMP-Efg1 pathway.

Oral candidiasis is the most common fungal infectious disease in the human oral cavity, and Candida albicans is the major pathogenic agent. Increasing drug resistance and the lack of new types of antifungals greatly increase the challenges for treating fungal infections. Targeting hyphal transition provides a promising strategy to inhibit the virulence of C. albicans and overcome drug resistance. This study aimed to investigate the effects and mechanisms of sigX-inducing peptide (XIP), a quorum-sensing signal peptide secreted by Streptococcus mutans, on C. albicans hyphal development and biofilm formation in vitro and oropharyngeal candidiasis in vivo. XIP significantly inhibited C. albicans yeast-to-hypha transition and biofilm formation in a dose-dependent manner from 0.01 to 0.1 µM. XIP significantly downregulated expression of genes from the Ras1-cAMP-Efg1 pathway (RAS1, CYR1, TPK2, EFG1 and UME6), a key pathway to regulate C. albicans hyphal development. Importantly, XIP reduced the levels of key molecules cAMP and ATP from this pathway, while the addition of exogenous cAMP and overexpression of RAS1 restored the hyphal development inhibited by XIP. XIP also lost its hyphal inhibitory effects on ras1Δ/Δ and efg1Δ/Δ strains. These results further confirmed that XIP inhibited hyphal development through downregulation of the Ras1-cAMP-Efg1 pathway. A murine oropharyngeal candidiasis model was employed to evaluate the therapeutic effects of XIP on oral candidiasis. XIP effectively reduced the infected epithelial area, fungal burden, hyphal invasion and inflammatory infiltrates. These results revealed the antifungal effects of XIP, and highlighted that XIP can be a potential antifungal peptide against C. albicans infection.

[Latest Findings on Polyketides/Non-ribosomal Peptides That Are Secondary Metabolites of Streptococcus mutans].

Dental caries is a chronic infectious disease that occurs in the hard tissue of teeth under the influence of multiple factors, among which bacteria being a key factor. Streptococcus mutans ( S. mutans) is considered a major pathogen that causes caries. Secondary metabolites, including bacteriocins and polyketides/non-ribosomal peptides, are a class of small-molecule compounds synthesized by S. mutans. To date, polyketides/non-ribosomal peptides identified in S. mutans include mutanobactin, mutanocyclin, and mutanofactin, which are synthesized by the mub, muc, and muf biosynthetic gene clusters, respectively. These polyketides/non-ribosomal peptides play important roles in bacterial inter-species competition, oxidative stress, and biofilm formation. In this review, we provided an overview of the synthesis, function and regulation of three polyketides/non-ribosomal peptides of S. mutans, including mutanobactin, mutanocyclin, and mutanofactin, aiming to provide new insights into the cariogenic mechanism of S. mutans and to promote the better management of dental caries.

The ComRS-SigX Pathway Regulates Natural Transformation in Streptococcus ferus.

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX), whose expression is induced by two types of peptide signals: CSP (competence stimulating peptide, encoded by comC) and XIP (sigX-inducing peptide, encoded by comS). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus, but not homologs of S. mutans blpRH (also known as comDE). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing sigX-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that cross talk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCE Natural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism.

Mussel-Inspired Caries Management Strategy: Constructing a Tribioactive Tooth Surface with Remineralization, Antibiofilm, and Anti-inflammation Activity.

Dental caries is a common chronic oral disease in humans resulting from tooth demineralization caused by acid production of bacterial plaque, which leads to the destruction of enamel and dentin and oral inflammation. However, it is still a challenge that the function of natural active ingredients in currently available oral care products is not comprehensive, especially the lack of remineralization. Here, inspired by the strong biological adhesion ability of mussels and ancient oral disease plant therapy, a multifunctional strategy is proposed to construct a bioactive tooth surface to treat dental caries. It has been demonstrated that the Turkish gall extract (TGE) can inhibit adhesion of cariogenic bacteria Streptococcus mutans and Actinomyces viscosus and destroy biofilms on the tooth surface. Meanwhile, TGE can reduce the expression of inflammatory factors. Notably, the TGE coating can induce the growth of hydroxyapatite (HAP) crystals in vivo and in vitro, recovering the enamel mechanical properties under normal oral conditions. MD simulations interpreted the adsorption mechanism by which the hydroxyl groups in TGE bind to phosphate group (PO43-) on the tooth surface, attracting calcium ions (Ca2+) as nucleation sites for remineralization. This work underlines the importance of TGE coating in remineralization, antibiofilm, and anti-inflammation activity as a promising strategy for dental caries.

ZccE, a P-type ATPase contributing to biofilm formation and competitiveness in Streptococcus mutans.

Most living organisms require zinc for survival; however, excessive amounts of this trace element can be toxic. Therefore, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent introduction of exogenous zinc ions, present a serious challenge for bacteria colonizing the oral cavity. Streptococcus mutans is considered one of the main bacterial pathobiont in dental caries. Here, we verified the role of a P-type ATPase ZccE as the main zinc-exporting transporter in S. mutans and delineated the effects of zinc toxification caused by zccE deletion in the physiology of this bacterium. The deletion of the gene zccE severely impaired the ability of S. mutans to grow under high zinc stress conditions. Intracellular metal quantification using inductively coupled plasma optical emission spectrometer revealed that the zccE mutant exhibited approximately two times higher zinc accumulation than the wild type when grown in the presence of a subinhibitory zinc concentration. Biofilm formation analysis revealed less single-strain biofilm formation and competitive weakness in the dual-species biofilm formed with Streptococcus sanguinis for zccE mutant under high zinc stress. The quantitive reverse transcription polymerase chain reaction test revealed decreased expressions of gtfB, gtfC, and nlmC in the mutant strain under excessive zinc treatment. Collectively, these findings suggest that ZccE plays an important role in the zinc detoxification of S. mutans and that zinc is a growth-limiting factor for S. mutans within the dental biofilm.

Improved Tumor Infiltration and Immunomodulation for Tumor Therapy: A Pathway Based on Tetrahedral Framework Nucleic Acids Coupled Bacterial Nanocells.

Growing evidence indicates that the tumor microenvironment (TME) can be combined with other therapeutic modalities, including cytotoxic chemotherapy and targeted therapies, to produce unanticipated results in oncology treatment. Here, we proposed a novel bacterial nanomaterial capable of targeting peritumoral biofilm and modulating TME. It was based on tetrahedral framework nucleic acids (T) that were chemically attached to aptamer AS1411 and 5-fluorouracil (AT5). Additionally, the oral pathogenic bacterium Streptococcus mutans (S.m) was employed as a biocarrier for synergetic biofilm targeting and immunomodulation. In this article, the effect of AT5-coupled S.m-derived nanocells (S.m-AT5) was investigated in vitro and in vivo. Due to bacteria aggregation in the tumor-specific biofilm, these nanocells released greater medication concentrations. Furthermore, they exerted an immunomodulatory effect by stimulating the maturation of dendritic cells (DCs) and regulation of T cells. This chemo-immunostimulation combination has a powerful antitumor impact. It may also be an advanced approach for boosting the survival rate of cancer patients.

ZccE is a Novel P-type ATPase That Protects Streptococcus mutans Against Zinc Intoxication.

Zinc is a trace metal that is essential to all forms of life, but that becomes toxic at high concentrations. Because it has both antimicrobial and anti-inflammatory properties and low toxicity to mammalian cells, zinc has been used as a therapeutic agent for centuries to treat a variety of infectious and non-infectious conditions. While the usefulness of zinc-based therapies in caries prevention is controversial, zinc is incorporated into toothpaste and mouthwash formulations to prevent gingivitis and halitosis. Despite this widespread use of zinc in oral healthcare, the mechanisms that allow Streptococcus mutans, a keystone pathogen in dental caries and prevalent etiological agent of infective endocarditis, to overcome zinc toxicity are largely unknown. Here, we discovered that S. mutans is inherently more tolerant to high zinc stress than all other species of streptococci tested, including commensal streptococci associated with oral health. Using a transcriptome approach, we uncovered several potential strategies utilized by S. mutans to overcome zinc toxicity. Among them, we identified a previously uncharacterized P-type ATPase transporter and cognate transcriptional regulator, which we named ZccE and ZccR respectively, as responsible for the remarkable high zinc tolerance of S. mutans. In addition to zinc, we found that ZccE, which was found to be unique to S. mutans strains, mediates tolerance to at least three additional metal ions, namely cadmium, cobalt, and copper. Loss of the ability to maintain zinc homeostasis when exposed to high zinc stress severely disturbed zinc:manganese ratios, leading to heightened peroxide sensitivity that was alleviated by manganese supplementation. Finally, we showed that the ability of the ΔzccE strain to stably colonize the rat tooth surface after topical zinc treatment was significantly impaired, providing proof of concept that ZccE and ZccR are suitable targets for the development of antimicrobial therapies specifically tailored to kill S. mutans.

Fluorescence lectin binding analysis of carbohydrate components in dental biofilms grown in situ in the presence or absence of sucrose.

Carbohydrate components, such as glycoconjugates and polysaccharides, are constituents of the dental biofilm matrix that play an important role in biofilm stability and virulence. Exopolysaccharides in Streptococcus mutans biofilms have been characterized extensively, but comparably little is known about the matrix carbohydrates in complex, in situ-grown dental biofilms. The present study employed fluorescence lectin binding analysis (FLBA) to investigate the abundance and spatial distribution of glycoconjugates/polysaccharides in biofilms (n = 306) from 10 participants, grown in situ with (SUC) and without (H2O) exposure to sucrose. Biofilms were stained with 10 fluorescently labeled lectins with different carbohydrate specificities (AAL, ABA, ASA, HPA, LEA, MNA-G, MPA, PSA, VGA and WGA) and analyzed by confocal microscopy and digital image analysis. Microbial composition was determined by 16S rRNA gene sequencing. With the exception of ABA, all lectins targeted considerable matrix biovolumes, ranging from 19.3% to 194.0% of the microbial biovolume in the biofilms, which illustrates a remarkable variety of carbohydrate compounds in in situ-grown dental biofilms. MNA-G, AAL, and ASA, specific for galactose, fucose, and mannose, respectively, stained the largest biovolumes. AAL and ASA biovolumes were increased in SUC biofilms, but the difference was not significant due to considerable biological variation. SUC biofilms were enriched in streptococci and showed reduced abundances of Neisseria and Haemophilus spp., but no significant correlations between lectin-stained biovolumes and bacterial abundance were observed. In conclusion, FLBA demonstrates the presence of a voluminous biofilm matrix comprising a variety of different carbohydrate components in complex, in situ-grown dental biofilms.

Streptococcus mutans e seu metabolismo a nível molecular no contexto ecológico da doença cárie / Streptococcus mutans and its metabolism at the molecular level in the ecological context of dental caries

Objetivo: Durante décadas, o Streptococcus mutans foi con-siderado o principal agente etiológico da doença cárie. Esta revisão apresentará seu histórico e metabolismo a nível molecular. Ao entender as vias metabólicas do S.mutans envolvidas no desenvolvimento de lesões cariosas, será possível desenvolver novos métodos de modulação de biofilmes no controle da doença cárie e elucidar a neces-sidade de continuar pesquisando essa bactéria. Revisão de literatura: Embora o S. mutans não constitua uma pro-porção significativa na colonização da microbiota bucal da dentição hígida, essa proporção aumenta quando há acidificação contínua do biofilme, associada ao excesso de carboidratos na dieta do hospedeiro. Isso ocorre devido a um conjunto de fatores de virulência, tais como, adesão, formação de biofilme, acidogenicidade, aciduricidade, atividades de proteases, produção de mutacinas e vias de transdução de sinal. Cada uma dessas propriedades, coordenadamente, alteram a ecologia do biofilme dental. Discussão: Ainda é relevante entender o metabolismo do S. mutans como microrganismo modelo em lesões cariosas devido a seus inúmeros fatores de virulência. Porém, no contexto da doença cárie como uma disbiose, estratégias terapêuticas antimicrobianas, mais especificamente anti-S.mutans, voltadas para a eliminação do microrganismo, po-dem não ser a chave do controle da doença cárie, enquanto a modulação do microbioma poderá se tornar o futuro das clínicas odontológicas. Conclusão: Biofilmes associados a doença cárie compreendem um ecossistema diverso, sugerindo uma etiologia polimicrobiana, porém, estudos futuros que visem à prospecção, ao desenvolvimento e à inter-relação do S. mutans com outros microrganismos e com o hospedeiro humano ainda são justificados a fim de desvendar a transição 'homeostase-disbiose'.

Aim: For decades, the Streptococcus mutans was consi-dered the main agent of caries. This review will show its history and metabolism at the molecular level. By understanding its metabolic pathways involved in the development of carious lesions, it can be possible to develop new methods of modulating biofilms in the control of caries, as well as to elucidate the need to continue researching this bacterium. Literature review: Although S. mutans does not constitute a significant proportion in the colonization of the oral microbiota of the sound dentition, its proportion increases when there is continuous acidification of the biofilm, asso-ciated with excess carbohydrates in the host diet. This is due to a set of virulence factors, such as adhesion, biofilm formation, acidogenicity, aciduricity, proteases activity, mutacins production and signal transduction pathways. Each of these properties coordinately alters the ecology of the dental biofilm. Discussion: It is still relevant to understand the metabolism of S. mutans as a model microorganism in carious lesions due to its numerous virulence factors. However, in the context of caries as a dysbiosis, antimicrobial therapeutic strategies, more specifically anti-S.mutans, aiming to eliminate the microorganism, may not be the key to caries control, and the microbiome modulation may become the future of dental clinics. Conclusion: Biofilms associated with caries disease comprise a diverse ecosystem, suggesting a polymicrobial etiolo-gy, however, future studies aimed at the prospection, development and interrelationship of S. mutans with other microorganisms and with the human host are still justified in order to unravel the 'homeos-tasis-dysbiosis' transition.

ClpX/P-Dependent Degradation of Novel Substrates in Streptococcus mutans.

Regulated proteolysis is where AAA+ ATPases (ClpX, ClpC, and ClpE) are coupled to a protease subunit (ClpP) to facilitate degradation of misfolded and native regulatory proteins in the cell. The process is intricately linked to protein quality control and homeostasis and modulates several biological processes. In streptococci, regulated proteolysis is vital to various functions, including virulence expression, competence development, bacteriocin production, biofilm formation, and stress responses. Among the various Clp ATPases, ClpX is the major one that recognizes specific amino acid residues in its substrates and delivers them to the ClpP proteolytic chamber for degradation. While multiple ClpX substrates have been identified in Escherichia coli and other bacteria, little is known about the identity of these substrates in streptococci. Here, we used a preliminary proteomic analysis to identify putative ClpX substrates using Streptococcus mutans as a model organism. SMU.961 is one such putative substrate where we identified the Glu-Lue-Gln (ELQ) motif at the C terminus that is recognized by ClpX/P. We identified several other proteins, including MecA, which also harbor ELQ and are degraded by ClpX/P. This is surprising since MecA is known to be degraded by ClpC/P in Bacillus subtilis; however, ClpX/P-mediated MecA degradation is unknown. We also identified Glu and Gln as the crucial residues for ClpX recognition. Our data indicate a species and perhaps strain-specific recognition of ELQ by streptococcal ClpX/P. At present, we do not know whether this species-dependent degradation by ClpX/P is unique to S. mutans, and we are currently examining the phenomenon in other pathogenic streptococci. IMPORTANCE ClpX/P is a major intracellular proteolytic complex that is responsible for protein quality control in the cell. ClpX, an AAA+ ATPase, distinguishes the potential substrates by recognizing short motifs at the C-terminal end of proteins and delivers the substrates for degradation by ClpP protease. The identity of these ClpX substrates, which varies greatly among bacteria, is known only for a few well-studied species. Here, we used Streptococcus mutans as a model organism to identify ClpX substrates. We found that a short motif of three residues is successfully recognized by ClpX/P. Interestingly, the motif is not present at the ultimate C-terminal end; rather it is present close to the end. This result suggests that streptococcal ClpX ATPase can recognize internal motifs.

Collagen Peptide in a Combinatorial Treatment with Lactobacillus rhamnosus Inhibits the Cariogenic Properties of Streptococcus mutans: An In Vitro Study.

Dental caries is caused by the formation of cariogenic biofilm, leading to localized areas of enamel demineralization. Streptococcus mutans, a cariogenic pathogen, has long been considered as a microbial etiology of dental caries. We hypothesized that an antagonistic approach using a prebiotic collagen peptide in combination with probiotic Lactobacillus rhamnosus would modulate the virulence of this cariogenic biofilm. In vitro S. mutans biofilms were formed on saliva-coated hydroxyapatite discs, and the inhibitory effect of a combination of L. rhamnosus and collagen peptide on S. mutans biofilms were evaluated using microbiological, biochemical, confocal imaging, and transcriptomic analyses. The combination of L. rhamnosus with collagen peptide altered acid production by S. mutans, significantly increasing culture pH at an early stage of biofilm formation. Moreover, the 3D architecture of the S. mutans biofilm was greatly compromised when it was in the presence of L. rhamnosus with collagen peptide, resulting in a significant reduction in exopolysaccharide with unstructured and mixed bacterial organization. The presence of L. rhamnosus with collagen peptide modulated the virulence potential of S. mutans via down-regulation of eno, ldh, and atpD corresponding to acid production and proton transportation, whereas aguD associated with alkali production was up-regulated. Gly-Pro-Hyp, a common tripeptide unit of collagen, consistently modulated the cariogenic potential of S. mutans by inhibiting acid production, similar to the bioactivity of a collagen peptide. It also enhanced the relative abundance of commensal streptococci (S. oralis) in a mixed-species biofilm by inhibiting S. mutans colonization and dome-like microcolony formation. This work demonstrates that food-derived synbiotics may offer a useful means of disrupting cariogenic communities and maintaining microbial homeostasis.

Comprehensive characterization of sortase A-dependent surface proteins in Streptococcus mutans.

Streptococcus mutans, a cariogenic pathogen, adheres to the tooth surface and forms a biofilm. Bacterial cell surface proteins are associated with adherence to substrates. Sortase A (SrtA) mediates the localization of proteins with an LPXTG motif-containing proteins to the cell surface by covalent binding to peptidoglycan. In S. mutans UA159, six SrtA-dependent proteins, SpaP, WapA, WapE, DexA, FruA, and GbpC, were identified. Although some of these proteins were characterized, a comprehensive analysis of the six proteins has not been reported. In this study, we constructed mutants deficient in each of these proteins and the SrtA-deficient mutant. The SrtA-deficient mutant showed drastically decreased binding to salivary components, biofilm formation, bacterial coaggregation activity, hydrophobicity, and cellular matrix binding (collagen type I, fibronectin, and laminin). The SpaP-deficient mutant showed significantly reduced binding to salivary components and partially increased coaggregation with Porphyromonas gingivalis, and decreased hydrophobicity, and collagen binding. The WapA-deficient mutant showed slightly decreased coaggregation with Fusobacterium nucleatum. Although the SrtA-deficient mutant showed drastically altered phenotypes, all SrtA-dependent protein-deficient mutants, except the SpaP-deficient mutant, did not show considerable alterations in binding to salivary components. These results indicate that the six proteins may coordinately contribute to these activities. In addition, using genomic data of 125 S. mutans strains, the amino acid sequences of each surface protein were compared and many variations were found among strains, which may affect the phenotype of cell surface proteins in S. mutans.

Elucidating the Role and Structure-Activity Relationships of the Streptococcus oligofermentans Competence-Stimulating Peptide.

Streptococcus oligofermentans is an early colonizer of the oral microbiome with documented bactericidal activity against the oral pathogen Streptococcus mutans. S. oligofermentans has been observed to possess the typical comABCDE competence regulon found within most oral streptococci; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon is yet to be explored. Herein, we have both confirmed the identity of the S. oligofermentans CSP and utilized a wide range of phenotypic assays to characterize its regulatory role in competence, biofilm formation, and hydrogen peroxide formation. To determine the importance of each amino acid residue in CSP/ComD binding, we performed systematic replacement of amino acid residues within the S. oligofermentans CSP and developed a luciferase-based reporter system to assess the ability of these mutated analogues to modulate the competence regulon. Additionally, we performed CD analysis on mutated CSP analogues to determine the correlation between the peptide secondary structure and QS activation. To further explore S. oligofermentans' potential as a biotherapeutic against S. mutans infection, lead QS activators and inhibitors were used in interspecies competition assays to assess the effect of QS modulation on interactions between these two species. Lastly, we have documented a lack of S. oligofermentans-induced cytotoxicity, highlighting the potential of this native flora as a biotherapeutic with minimal health risks.

Effects of cigarette smoking on the growth of Streptococcus mutans biofilms: An in vitro study.

The increased incidence of dental caries by cigarette smoking (CS) has been widely reported in epidemiological studies, but the relationship between CS and cariogenic biofilm growth has been rarely studied. This study aims to investigate the effects of CS exposure on the growth and virulence of Streptococcus mutans biofilms (S. mutans). Briefly, S. mutans biofilms were formed on saliva-coated hydroxyapatite disks, which were exposed to CS 1, 3, and 6 times per day, respectively. In addition, S. mutans biofilms without CS exposure were considered as the control group. Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides (IPSs) were analyzed and confocal laser scanning microscopy (CLSM) images of 74-h-old S. mutans biofilms were obtained. The lowest accumulation of biofilms and EPSs were detected in the 6 times/day CS exposure group compared with those of the control group and other CS exposure groups in 74-h-old S. mutans biofilms. CLSM also revealed the lowest bacterial count (live and dead cells) and EPSs biovolume in the six times/day CS exposure group in 74-h-old S. mutans biofilms. CS exposure inhibited the growth of S. mutans biofilm in vitro study, the anti-cariogenic biofilm formation was enhanced with a dose (frequency)-dependent at which frequency has more influence in the present findings.

Bitter Taste Receptor T2R14 Modulates Gram-Positive Bacterial Internalization and Survival in Gingival Epithelial Cells.

Bitter-taste receptors (T2Rs) have emerged as key players in host-pathogen interactions and important modulators of oral innate immunity. Previously, we reported that T2R14 is expressed in gingival epithelial cells (GECs) and interacts with competence stimulating peptides (CSPs) secreted by the cariogenic Streptococcus mutans. The underlying mechanisms of the innate immune responses and physiological effects of T2R14 on Gram-positive bacteria are not well characterized. In this study, we examined the role of T2R14 in internalization and growth inhibitory effects on Gram-positive bacteria, namely Staphylococcus aureus and S. mutans. We utilized CRISPR-Cas9 T2R14 knockdown (KD) GECs as the study model to address these key physiological mechanisms. Our data reveal that the internalization of S. aureus is significantly decreased, while the internalization of S. mutans remains unaffected upon knockdown of T2R14 in GECs. Surprisingly, GECs primed with S. mutans CSP-1 resulted in an inhibition of growth for S. aureus, but not for S. mutans. The GECs infected with S. aureus induced T2R14-dependent human ß-defensin-2 (hBD-2) secretion; however, S. mutans-infected GECs did not induce hBD-2 secretion, but induced T2R14 dependent IL-8 secretion. Interestingly, our results show that T2R14 KD affects the cytoskeletal reorganization in GECs, thereby inhibiting S. aureus internalization. Our study highlights the distinct mechanisms and a direct role of T2R14 in influencing physiological responses to Gram-positive bacteria in the oral cavity.