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Protein acetylation is a common and reversible posttranslational modification tightly governed by protein acetyltransferases and deacetylases crucial for various biological processes in both eukaryotes and prokaryotes. Although recent studies have characterized many acetyltransferases in diverse bacterial species, only a few protein deacetylases have been identified in prokaryotes, perhaps in part due to their limited sequence homology. In this study, we identified YkuR, encoded by smu_318 , as a unique protein deacetylase in Streptococcus mutans . Through protein acetylome analysis, we demonstrated that the deletion of ykuR significantly upregulated protein acetylation levels, affecting key enzymes in translation processes and metabolic pathways, including starch and sucrose metabolism, glycolysis/gluconeogenesis, and biofilm formation. In particular, YkuR modulated extracellular polysaccharide synthesis and biofilm formation through the direct deacetylation of glucosyltransferases (Gtfs) in the presence of NAD + . Intriguingly, YkuR can be acetylated in a nonenzymatic manner, which then negatively regulated its deacetylase activity, suggesting the presence of a self-regulatory mechanism. Moreover, in vivo studies further demonstrated that the deletion of ykuR attenuated the cariogenicity of S. mutans in the rat caries model, substantiating its involvement in the pathogenesis of dental caries. Therefore, our study revealed a unique regulatory mechanism mediated by YkuR through protein deacetylation that regulates the physiology and pathogenicity of S. mutans ., Competing Interests: Competing interests statement:The authors declare no competing interest.

Numerous cellular processes are regulated in response to the metabolic state of the cell, and one such regulatory mechanism involves lysine acetylation. Lysine acetylation has been proven to play an important role in the virulence of Streptococcus mutans, a major cariogenic bacterial species. S. mutans' glucosyltransferases (Gtfs) are responsible for synthesizing extracellular polysaccharides (EPS) and contributing to biofilm formation. One of the most common nonsteroidal anti-inflammatory drugs is acetylsalicylic acid (ASA), which can acetylate proteins through a nonenzymatic transacetylation reaction. Herein, we investigated the inhibitory effects of ASA on S. mutans. ASA treatment was observed to impede the growth of S. mutans, leading to a reduction in the production of water-insoluble EPS and the formation of biofilm. Moreover, ASA decreased the enzyme activity of Gtfs while increasing the protein acetylation level. The in vivo anticaries efficacy of ASA has further been proved using the rat caries model. In conclusion, ASA as an acetylation agent attenuated the cariogenic virulence of S. mutans, suggesting the potential value of protein acetylation on antimicrobial and anti-biofilm applications to S. mutans., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dental caries is the most common chronic infectious disease around the world and disproportionately affects the marginalized socioeconomic group. Streptococcus mutans , considered a primary etiological agent of caries, depends on the coordinated physiological response to tolerate the oxidative stress generated by commensal species within dental plaque, which is a critical aspect of its pathogenicity. Here, we identified and characterized a novel tetracycline repressor family regulator, SMU_1361c, which appears to be acquired by the bacteria via horizontal gene transfer. Surprisingly, smu_1361c functions as a negative transcriptional regulator to regulate gene expression outside its operon and is involved in the oxidative stress response of S. mutans . The smu_1361c overexpression strain UA159/pDL278- 1361c was more susceptible to oxidative stress and less competitive against hydrogen peroxide generated by commensal species Streptococcus gordonii and Streptococcus sanguinis . Transcriptomics analysis revealed that smu_1361c overexpression resulted in the significant downregulation of 22 genes, mainly belonging to three gene clusters responsible for the oxidative stress response. The conversed DNA binding motif of SMU_1361c was determined by electrophoretic mobility shift and DNase I footprinting assay with purified SMU_1361c protein; therefore, smu_1361c is directly involved in gene transcription related to the oxidative stress response. Crucially, our finding provides a new understanding of how S. mutans deals with the oxidative stress that is required for pathogenesis and will facilitate the development of new and improved therapeutic approaches for dental caries.IMPORTANCE Streptococcus mutans is the major organism associated with the development of dental caries, which globally is the most common chronic disease. To persist and survive in biofilms, S. mutans must compete with commensal species that occupy the same ecological niche. Here, we uncover a novel molecular mechanism of how tetracycline repressor family regulator smu_1361c is involved in the oxidative stress response through transcriptomics analysis, electrophoretic mobility shift assay, and DNase I footprinting assay. Furthermore, we demonstrated that smu_1361c mediates S. mutans sensitivity to oxidative stress and competitiveness with commensal streptococci. Therefore, this study has revealed a previously unknown regulation between smu_1361c and genes outside its operon and demonstrated the importance of smu _ 1361c in the oxidative stress response and the fitness of S. mutans within the plaque biofilms, which can be exploited as a new therapy to modulate ecological homeostasis and prevent dental caries., Competing Interests: The authors declare no conflict of interest.

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., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Streptococcus mutans is considered to be a major causative agent of dental caries. VicRK is a two-component signal transduction system (TCSTS) of S. mutans, which can regulate the virulence of S. mutans, such as biofilm formation, exopolysaccharide production, acid production, and acid resistance. Meanwhile, it can also regulate the production of mutacins (nlmC) through the TCSTS ComDE. In this study, we found that the vicR-overexpressing strain was more likely to aggregate to form cell clusters, leading to the formation of abnormal biofilm; the overexpression of vicR increased the length of the chain of S. mutans. Furthermore, the expression of the mutacins in the vicR overexpression strain was increased under aerobic conditions. Compared with the control strain and the parental strain, the vicR overexpression strain was more competitive against Streptococcus gordonii. But there was no significant difference against Streptococcus sanguinis. In clinical strains, the expression level of vicR was positively correlated with their competitive ability against S. gordonii. Transcriptional profiling revealed 24 significantly upregulated genes in the vicR-overexpressing strain, including nlmA, nlmB, nlmC, and nlmD encoding mutacins. Electrophoretic mobility shift assays and DNase I footprinting assays confirmed that VicR can directly bind to the promoter sequence of nlmD. Taken together, our findings further demonstrate that VicRK, an important TCSTS of S. mutans, is involved in S. mutans cell morphology and biofilm formation. VicRK regulates the production of more mutacins in S. mutans in response to oxygen stimulation. VicR can bind to the promoter sequence of nlmD, thereby directly regulating the production of mutacins NlmD., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dental caries is a chronic progressive disease, which destructs dental hard tissues under the influence of multiple factors, mainly bacteria. Streptococcus mutans is the main cariogenic bacteria. However, its cariogenic virulence is affected by environmental stress such as oxidative stress, nutrient deficiency, and low pH to some extent. Oxidative stress is one of the main stresses that S. mutans faces in oral cavity. But there are a variety of protective molecules to resist oxidative stress in S. mutans, including superoxide dismutase, nicotinamide adenine dinucleotide oxidase, Dps-like peroxide resistance protein, alkyl-hydrogen peroxide reductase, thioredoxin, glutamate-reducing protein system, and some metabolic substances. Additionally, some transcriptional regulatory factors (SloR, PerR, Rex, Spx, etc.) and two-component systems are also closely related to oxidative stress adaptation by modulating the expression of protective molecules. This review summarizes the research progress of protective molecules and regulatory mechanisms (mainly transcription factors) of oxidative stress adaptation of S. mutans., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Lysine acetylation, a ubiquitous and dynamic regulatory posttranslational modification (PTM), affects hundreds of proteins across all domains of life. In bacteria, lysine acetylation can be found in many essential pathways, and it is also crucial for bacterial virulence. However, the biological significance of lysine acetylation events to bacterial virulence factors remains poorly characterized. In Streptococcus mutans, the acetylome profiles help identify several lysine acetylation sites of lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactic acid, causing the deterioration of teeth. We investigated the regulatory mechanism of LDH acetylation and characterized the effect of LDH acetylation on its function. We overexpressed the 15 Gcn5 N -acetyltransferases (GNAT) family members in S. mutans and showed that the acetyltransferase ActA impaired its acidogenicity by acetylating LDH. Additionally, enzymatic acetyltransferase reactions demonstrated that purified ActA could acetylate LDH in vitro , and 10 potential lysine acetylation sites of LDH were identified by mass spectrometry, 70% of which were also detected in vivo . We further demonstrated that the lysine acetylation of LDH inhibited its enzymatic activity, and a subsequent rat caries model showed that ActA impaired the cariogenicity of S. mutans. Collectively, we demonstrated that ActA, the first identified and characterized acetyltransferase in S. mutans, acetylated the LDH enzymatically and inhibited its enzymatic activity, thereby providing a starting point for the further analysis of the biological significance of lysine acetylation in the virulence of S. mutans. IMPORTANCE Lysine acetylation, a dynamic regulatory posttranslational modification, remains poorly characterized in bacteria. Hundreds of proteins have been identified to be acetylated in bacteria, with advances made in acetylome analyses. However, the regulatory mechanisms and functional significance of the majority of these acetylated proteins remain unclear. We analyzed the acetylome profiles of Streptococcus mutans and found that lactate dehydrogenase (LDH) contains several lysine acetylation sites. We also demonstrated that the acetyltransferase ActA, a member of the Gcn5 N -acetyltransferases (GNAT) family in S. mutans, acetylated LDH, inhibited its enzymatic ability to catalyze the conversion of pyruvate to lactic acid, and impaired its cariogenicity in a rat caries model. Therefore, LDH acetylation might be a potential target that can be exploited in the design of novel therapeutics to prevent dental caries.

Streptococcus mutans is known as an important oral pathogen causing dental caries, a widespread oral infectious disease. S. mutans synthesize exopolysaccharide (EPS) using glucosyltransferases (Gtfs), resulting in biofilm formation on the tooth surface. Bacterial cells in the biofilms become strongly resistant to a harsh environment, such as antibiotics and host defense mechanisms, making biofilm-based infections difficult to eliminate. Discovering novel antibiofilm agents, especially from natural products, helps to develop effective strategies against this kind of diseases. The present study investigated the inhibitory effect of shikimic acid (SA), one abundant compound derived from Illicium verum extract, on the biofilm formation of S. mutans. We found SA can reduce the EPS synthesized by this oral pathogen and modulate the transcription of biofilm formation related genes, leading to fewer bacterial cells in its biofilm. SA also interacted with cell membrane and membrane proteins, causing damage to bacterial cells. Ex vivo testing of biofilm formation on bovine teeth showed SA strongly decreased the number of S. mutans cells and the number of EPS accumulated on dental enamel surfaces. Moreover, SA exhibits almost no toxicity to human oral cells evaluated by in vitro biocompatibility assay. In conclusion, shikimic acid exhibits remarkable antibiofilm activity against S. mutans and has the potential to be further developed as a novel anticaries agent. IMPORTANCE Natural products are an important and cost-effective source for screening antimicrobial agents. Here, we identified one compound, shikimic acid, from Illicium verum extract, exhibiting antimicrobial activity against S. mutans proliferation. It also inhibits biofilm formation of this bacteria through decreasing Gtf expression and EPS synthesis. Furthermore, this compound exhibits no significant cytotoxicity at its MIC against S. mutans, providing evidence for its clinical application.

Streptococcus mutans is a primary cariogenic pathogen in humans. Arginine metabolism is required for bacterial growth. In S. mutans, however, the involvement of transcription factors in regulating arginine metabolism is unclear. The purpose of this study was to investigate the function and mechanism of ArgR family transcription factors in S. mutans. Here, we identified an ArgR (arginine repressor) family transcription factor named AhrC, which negatively regulates arginine biosynthesis and biofilm formation in S. mutans. The ahrC in-frame deletion strain exhibited slow growth and significantly increased intracellular arginine content. The strain overexpressing ahrC showed reduced intracellular arginine content, decreased biofilm biomass, reduced production of water-insoluble exopolysaccharides (EPS), and different biofilm structures. Furthermore, global gene expression profiles revealed differential expression levels of 233 genes in the ahrC -deficient strain, among which genes related to arginine biosynthesis ( argJ , argB , argC , argD , argF , argG , argH ) were significantly upregulated. In the ahrC overexpression strain, there are 89 differentially expressed genes, mostly related to arginine biosynthesis. The conserved DNA patterns bound by AhrC were identified by electrophoretic mobility shift assay (EMSA) and DNase I footprinting. In addition, the analysis of β-galactosidase activity showed that AhrC acted as a negative regulator. Taken together, our findings suggest that AhrC is an important transcription factor that regulates arginine biosynthesis gene expression and biofilm formation in S. mutans. These findings add new aspects to the complexity of regulating the expression of genes involved in arginine biosynthesis and biofilm formation in S. mutans. IMPORTANCE Arginine metabolism is essential for bacterial growth. The regulation of intracellular arginine metabolism in Streptococcus mutans, one of the major pathogens of dental caries, is unclear. In this study, we found that the transcription factor AhrC can directly and negatively regulate the expression of N -acetyl-gamma-glutamyl-phosphate reductase ( argC ), thus regulating arginine biosynthesis in S. mutans. In addition, the ahrC overexpression strain exhibited a significant decrease in biofilm and water-insoluble extracellular polysaccharides (EPS). This study adds new support to our understanding of the regulation of intracellular arginine metabolism in S. mutans.

Early childhood caries (ECC) is a significant chronic disease of childhood and a rising public health burden worldwide. ECC may cause a higher risk of new caries lesions in both primary and permanent dentition, affecting lifelong oral health. The occurrence of ECC has been closely related to the core microbiome change in the oral cavity, which may be influenced by diet habits, oral health management, fluoride use, and dental manipulations. So, it is essential to improve parental oral health and awareness of health care, to establish a dental home at the early stage of childhood, and make an individualized caries management plan. Dental interventions according to the minimally invasive concept should be carried out to treat dental caries. This expert consensus mainly discusses the etiology of ECC, caries-risk assessment of children, prevention and treatment plan of ECC, aiming to achieve lifelong oral health., (© 2022. The Author(s).)

Oral bacteria directly affect the disease status of dental caries and periodontal diseases. The dynamic oral microbiota cooperates with the host to reflect the information and status of immunity and metabolism through two-way communication along the oral cavity and the systemic organs. The oral cavity is one of the most important interaction windows between the human body and the environment. The microenvironment at different sites in the oral cavity has different microbial compositions and is regulated by complex signaling, hosts, and external environmental factors. These processes may affect or reflect human health because certain health states seem to be related to the composition of oral bacteria, and the destruction of the microbial community is related to systemic diseases. In this review, we discussed emerging and exciting evidence of complex and important connections between the oral microbes and multiple human systemic diseases, and the possible contribution of the oral microorganisms to systemic diseases. This review aims to enhance the interest to oral microbes on the whole human body, and also improve clinician's understanding of the role of oral microbes in systemic diseases. Microbial research in dentistry potentially enhances our knowledge of the pathogenic mechanisms of oral diseases, and at the same time, continuous advances in this frontier field may lead to a tangible impact on human health., (© 2022. The Author(s).)

Diadenosine-5',5'''-P1, P4-tetraphosphate (Ap4A) is a second messenger playing a crucial role in various life activities of bacteria. The increase of Ap4A expression is pleiotropic, resulting in an impairment in the formation of biofilm and other physiological functions in some bacteria. However, Ap4A function in Streptococcus mutans, an important pathogen related to dental caries, remains unknown. In this work, the Ap4A hydrolase, YqeK, was identified and characterized in S. mutans. Then, the effects of yqeK deletion on the growth, biofilm formation, and exopolysaccharide (EPS) quantification in S. mutans were determined by the assessment of the growth curve, crystal violet, and anthrone-sulfuric acid, respectively, and visualized by microscopy. The results showed that the in-frame deletion of the yqeK gene in S. mutans UA159 led to an increase in Ap4A levels, lag phase in the early growth, as well as decrease in biofilm formation and water-insoluble exopolysaccharide production. Global gene expression profile showed that the expression of 88 genes was changed in the yqeK mutant, and among these, 42 were upregulated and 46 were downregulated when compared with the wild-type S. mutans UA159. Upregulated genes were mainly involved in post-translational modification, protein turnover, and chaperones, while downregulated genes were mainly involved in carbohydrate transport and metabolism. Important virulence genes related to biofilms, such as gtfB, gtfC, and gbpC, were also significantly downregulated. In conclusion, these results indicated that YqeK affected the formation of biofilms and the expression of biofilm-related genes in S. mutans., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dental caries is one of the most prevalent and costly biofilm-dependent oral infectious diseases affecting most of the world's population. Streptococcus mutans, a major extracellular polymeric substance (EPS) producing bacteria in dental plaque, plays a vital role in human dental caries. EPS acts as the framework of dental plaque and promotes bacterial adhesion, cohesion, and environmental stress resistance and hinders the diffusion of nutrients and metabolic products. Since EPS is critical for biofilm lifestyle and virulence of cariogenic bacteria, EPS disruption could be a potential strategy to prevent caries. This review sought to summarize potential strategies to inhibit S. mutans biofilms through EPS disruption. The signal network intervention has a positive effect on S. mutans biofilm disruption, which could be achieved by using cyclic dimeric G/AMP inhibitors, quorum sensing inhibitors, and diffusible signal factors. Besides the enzyme degradation of exopolysaccharides, extracellular DNA, and proteins, other novel strategies, such as nanoparticles and phage therapy, could also promote EPS matrix disruption., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Zinc (Zn 2+ ) is an essential divalent trace metal for living cells. Intracellular zinc homeostasis is critical to the survival and virulence of bacteria. Thus, the frequent fluctuations of salivary zinc, caused by the low physiological level and the frequent exogenous zinc introduction, present a serious challenge for bacteria colonizing the oral cavity. However, the regulation strategies to keep intracellular Zn 2+ homeostasis in Streptococcus mutans, an important causative pathogen of dental caries, are unknown. Because zinc uptake is primarily mediated by an ATP-binding ABC transporter AdcABC in Streptococcus strains, we examined the function of AdcABC and transcription factor AdcR in S. mutans in this study. The results demonstrated that deletion of either adcA or adcCB gene impaired the growth but enhanced the extracellular polymeric matrix production in S. mutans, both of which could be relieved after excessive Zn 2+ supplementation. Using RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction examination, LacZ-reporter studies, and electrophoretic mobility shift assay, we showed that a MarR (multiple antibiotic resistance regulator) family transcription factor, AdcR, negatively regulates the expression of the genes adcR, adcC, adcB, and adcA by acting on the adcRCB and adcA promoters in response to Zn 2+ concentration in their environmental niches. The deletion of adcR increases the sensitivity of S. mutans to excessive Zn 2+ supply. Taken together, our findings suggest that Adc regulon, which consists of a Zn 2+ uptake transporter AdcCBA and a Zn 2+ -responsive repressor AdcR, plays a prominent role in the maintenance of intracellular zinc homeostasis of S. mutans., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dental caries is one of the most prevalent and costly biofilm-associated infectious diseases affecting most of the world's population. In particular, dental caries is driven by dysbiosis of the dental biofilm adherent to the enamel surface. Specific types of acid-producing bacteria, especially Streptococcus mutans , colonize the dental surface and cause damage to the hard tooth structure in the presence of fermentable carbohydrates. Streptococcus mutans has been established as the major cariogenic pathogen responsible for human dental caries, with a high ability to form biofilms. The exopolysaccharide (EPS) matrix, mainly contributed by S. mutans , has been considered as a virulence determinant of cariogenic biofilm. As EPS is an important virulence factor, targeting EPS metabolism could be useful in preventing cariogenic biofilm formation. This review summarizes plausible strategies targeting S. mutans biofilms by degrading EPS structure, inhibiting EPS production, and disturbing the EPS metabolism-related gene expression and regulatory systems.

Nonnutritive sweeteners (NNSs) are sugar substitutes widely used to reduce the negative health effects of excessive sugar consumption. Dental caries, one of the most prevalent chronic diseases globally, results from a pathogenic biofilm with microecological imbalance and frequent exposure to sugars. Some research has shown that certain NNSs possess less cariogenic potential than sucrose, indicating their putative effect on oral microbiome. To uncover the alterations of acidogenic pathogens and alkali-generating commensals, as well as the biofilm cariogenic potential under the influence of NNSs, we selected four common NNSs (acesulfame-K, aspartame, saccharin, and sucralose) and established single-, dual-, and multispecies in vitro culture model to assess their effects on Streptococcus mutans ( S. mutans ) and/or Streptococcus sanguinis ( S. sanguinis ) compared to sucrose with the same sweetness. The results showed that NNSs significantly suppressed the planktonic growth, acid production, and biofilm formation of S. mutans or S. sanguinis compared with sucrose in single-species cultures. Additionally, decreased S. mutans / S. sanguinis ratio, less EPS generation, and higher pH value were observed in dual-species and saliva-derived multispecies biofilms with supplementary NNSs. Collectively, this study demonstrates that NNSs inhibit the cariogenic potential of biofilms by maintaining microbial equilibrium, thus having a promising prospect as anticaries agents., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Jianhui Zhu et al.)

Objective: The current study aimed to assess the antimicrobial activity of ursolic acid (UA) against multi-species biofilms formed by Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii, as well as to measure its biocompatibility., Methods: Crystal violet staining, CFU counting, CCK-8 assays and scanning electron microscopy (SEM) were applied to investigate the effect of UA on multi-species biofilms. UA's effect on exopolysaccharides (EPS) production was measured using confocal laser scanning microscopy (CLSM) and the anthrone-sulfuric acid method. Fluorescent in situ hybridization (FISH) was applied to visualize and quantify the microbial composition of multi-species biofilms. Quantitative real-time PCR (qRT-PCR) was used to measure the expression of virulence genes of S. mutans, S. sanguinis, and S. gordonii under UA treatment. Moreover, CCK-8 assays were performed to evaluate its cytotoxicity against human oral keratinocytes (HOKs) and human gingival epithelial cells (HGEs)., Results: The results showed that UA had significant antimicrobial activity against common oral streptococci. UA also reduced the EPS synthesis of oral streptococci and suppressed gtf genes' expression. In addition, UA reduced the proportion of S. mutans in multi-species biofilms. Besides, UA had low cytotoxicity against HOKs and HGEs., Conclusions: UA exhibited antibiofilm activity against oral pathogenic bacteria and had the potential to be used in dental caries treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ligustrum robustum (Roxb.) Blume is utilized as a traditional Chinese herbal tea with various health benefits and protective effects. Streptococcus mutans is an important cariogenic oral bacteria species. The present study aimed to assess the influence of Ligustrum robustum extract (LRE) on the biofilm formation of S. mutans and the mechanism of its action, as well as to identify its chemical components. For chemical identification, HPLC-MS and nuclear magnetic resonance were applied and four identified phytochemicals were reported (Ligurobustoside B, Ligurobustoside N, Ligurobustoside J, and Ligurobustoside C). The dose-dependent (0.5 to 2.0 μg/μL) antimicrobial toxicity of LRE against S. mutans biofilm formation and exopolysaccharide (EPS) synthesis was evaluated by confocal laser scanning microscopy (CLSM), Crystal violet stain, and CFU counting. The microstructure of S. mutans biofilm treated with LRE was investigated both on glass coverslips and ex vivo bovine dental enamel by scanning electron microscopy (SEM). Moreover, LRE downregulated the expression of S. mutans glucosyltransferase-encoding genes gtfB, gtfC, and gtfD, and the quorum sensing (QS) factors comD and comE, suggesting its toxic mechanism. In addition, the result of CCK-8 test on human oral cells revealed an acceptable biocompatibility of LRE. These findings indicated the possible application of this daily consumed herbal tea for caries prevention., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: The present study aimed to evaluate the effect of Rhodiola rosea extract (RE) on Streptococcus mutans biofilm formation and the relevant mechanism of its action., Methods: The effect of RE on the biofilm formation and extracellular polysaccharides (EPS) synthesis of S. mutans was assessed by confocal laser scanning microscopy (CLSM), crystal violet staining and CFU counting method. Scanning electron microscopy (SEM) was applied to observe the surface morphology of S. mutans biofilms formed on glass coverslips and dental enamel. To study the relevant mechanism, quantitative real time PCR (qRT-PCR) and zymogram assay were applied to measure the expression of virulence genes and the enzymatic activity of glucosyltransferases (Gtfs) under the treatment of RE. The CCK-8 assay was also performed on macrophages (RAWs) and human oral keratinocytes (HOKs) in order to evaluate its biocompatibility., Results: As a result, RE inhibited the biofilm formation and EPS synthesis of S. mutans. RE also suppressed the expression of gtf genes and quorum sensing (QS) system as well as the enzymatic activity of Gtf proteins. Moreover, RE exhibited a good biocompatibility to human cells., Conclusions: This study provides the evidence for RE as a novel anti-biofilm agent for clinical use., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: Cedrus deodara (Roxb. ex D.Don) G. Don is applied as anti-inflammatory and anti-infection agents in folklore medicine., Aim of the Study: The present study aimed to assess the antimicrobial activity of Cedrus deodara (Roxb. ex D.Don) G. Don extract (CDE) against Streptococcus mutans biofilm formation and its biocompatibility, as well as to identify its chemical components., Materials and Methods: Confocal laser scanning microscopy (CLSM), crystal violet staining, and CFU counting assay were applied to investigate the effect of CDE on S. mutans biofilm formation and extracellular polysaccharides (EPS) synthesis. The microstructure of S. mutans biofilms formed on glass coverslips and bovine enamel treated with CDE was observed by scanning electron microscopy (SEM). qRT-PCR was used to measure the expression of virulence genes gtfB, gtfC, and gtfD, and zymogram assay was performed to investigate the enzymatic activity of Gtfs. Moreover, HPLC-MS and NMR were applied to identify its chemical components. CCK-8 assay was also performed on human oral cells to evaluate its biocompatibility., Results: Under the treatment of CDE, S. mutans formed less biofilm on both coverslips and enamel surfaces and synthesized less EPS. Moreover, CDE downregulated the expression of gtf genes and inhibited the enzymatic activity of Gtfs. According to HPLC-MS and NMR results, molecular structures of six main compounds in CDE were identified. CDE also has a good biocompatibility., Conclusions: CDE exhibits inhibitory activity against S. mutans and a good biocompatibility. It has the potential to be developed as anti-caries agents for clinical use., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacterial cell division is initiated by tubulin homologue FtsZ that assembles into a ring structure at mid-cell to facilitate cytokinesis. EzrA has been identified to be implicated in FtsZ-ring dynamics and cell wall biosynthesis during cell division of Bacillus subtilis and Staphylococcus aureus, the model rod and cocci. However, its role in pathogenic streptococci remains largely unknown. Here, the role of EzrA was investigated in Streptococcus mutans, the primary etiological agent of human dental caries, by constructing an ezrA in-frame deletion mutant. Our data showed that the ezrA mutant was slow-growing with a shortened length and extended width round cell shape compared to the wild type, indicating a delay in cell division with abnormalities of peptidoglycan biosynthesis. Additionally, FtsZ irregularly localized in dividing ezrA mutant cells forming angled division planes, potentially contributing to an aberrant cell shape. Furthermore, investigation using single-species cariogenic biofilm model revealed that deletion of ezrA resulted in defective biofilm formation with less extracellular polysaccharides and altered three-dimensional biofilm architecture. Unexpectedly, in a dual-species ecological model, the ezrA mutant exhibited substantially lower tolerance for H 2 O 2 and reduced competitiveness against one commensal species, Streptococcus sanguinis. Taken together, these results demonstrate that EzrA plays a key role in regulating cell division and maintaining a normal morphology in S. mutans and is required for its robust biofilm formation/interspecies competition. Therefore, EzrA protein represents a potential therapeutic target in the development of drugs controlling dental caries and other biofilm-related diseases., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Streptococcus mutans is considered the predominant etiological agent of dental caries with the ability to form biofilm on the tooth surface. And, its abilities to obtain nutrients and metabolize fermentable dietary carbohydrates to produce acids contribute to its pathogenicity. The responses of S. mutans to environmental stresses are essential for its survival and role in cariogenesis. The VicRK system is one of the 13 putative TCS of S. mutans . The conserved functions of the VicRK signal transduction system is the key regulator of bacterial oxidative stress responses, acidification, cell wall metabolism, and biofilm formation. In this paper, it was discussed how the VicRK system regulates S. mutans virulence including bacterial physiological function, operon structure, signal transduction, and even post-transcriptional control in its regulon. Thus, this emerging subspecialty of the VicRK regulatory networks in S. mutans may strengthen our understandings aimed at providing a basis for the prevention of dental caries.

Oral streptococci are among the most abundant genera present in the oral cavity. They are usually the first colonizers of oral surfaces and they develop extensive microbial interactions, playing a fundamental role in the pathogenesis of oral diseases such as dental caries and periodontitis. In addition to physical adherence, streptococcal cells also exchange messages with cells from another Streptococcus spp. and other microorganisms in the form of metabolites and signaling molecules. In this review, we focused on these intrageneric and intergeneric interactions, and their association with oral diseases.

Objective: The aim of this study was to analyze the characteristics of the clustered regularly interspaced short palindromic repeats (CRISPR) sites in 45 clinical Streptococcus mutans strains and their relationship to the clinical manifestations of early childhood caries (ECC)., Methods: Forty-five S. mutans strains were isolated from the plaque samples taken from sixty-three children. CRISPR sites were sequenced and BLAST was used to compare these sites to those in the CRISPRTarget database. The association between the distribution of CRISPR sites and the manifestation of caries was analyzed by Chi-Square test. Further, biofilm formation (by crystal violet staining) and the synthesis of polysaccharide (by anthrone-sulfuric method) of all clinical isolated S. mutans strains with both CRISPR sites and no CRISPR site were comapared. Finally, acidogenicity and acidurity of two typical strains were determined using pH drop and acid tolerance assays. Biofilm formation and EPS synthesis by two typical strains were compared by 3D CLSM (Confocal Laser Scanning Microscope) assays and the expression of gtf genes were evaluated using qPCR., Results: We found that most of the spacers in the clinical S. mutans strains were derived from Streptococcus phages APCM01 and M102. The number of CRISPR sites in these strains was associated with the clinical manifestations of ECC. Moreover, we found that the biofilm formation and EPS synthesis ability of the S. mutans strains with both CRISPR sites was significant improved., Conclusions: An association was found between the distribution of CRISPR sites and the clinical manifestations of caries. The CRISPR sites might contribute to the cariogenic potential of S. mutans., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Dental plaque biofilms are responsible for numerous chronic oral infections and cause a severe health burden. Many of these infections cannot be eliminated, as the bacteria in the biofilms are resistant to the host's immune defenses and antibiotics. There is a critical need to develop new strategies to control biofilm-based infections. Biofilm formation in Streptococcus mutans is promoted by major virulence factors known as glucosyltransferases (Gtfs), which synthesize adhesive extracellular polysaccharides (EPS). The current study was designed to identify novel molecules that target Gtfs, thereby inhibiting S. mutans biofilm formation and having the potential to prevent dental caries. Structure-based virtual screening of approximately 150,000 commercially available compounds against the crystal structure of the glucosyltransferase domain of the GtfC protein from S. mutans resulted in the identification of a quinoxaline derivative, 2-(4-methoxyphenyl)-N-(3-{[2-(4-methoxyphenyl)ethyl]imino}-1,4-dihydro-2-quinoxalinylidene)ethanamine, as a potential Gtf inhibitor. In vitro assays showed that the compound was capable of inhibiting EPS synthesis and biofilm formation in S. mutans by selectively antagonizing Gtfs instead of by killing the bacteria directly. Moreover, the in vivo anti-caries efficacy of the compound was evaluated in a rat model. We found that the compound significantly reduced the incidence and severity of smooth and sulcal-surface caries in vivo with a concomitant reduction in the percentage of S. mutans in the animals' dental plaque (P < 0.05). Taken together, these results represent the first description of a compound that targets Gtfs and that has the capacity to inhibit biofilm formation and the cariogenicity of S. mutans., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Streptococcus mutans (S. mutans) phage, as one of the principal pathogenic bacteria of dental caries, is a main cause of the formation and development of dental caries due to its overproliferation in dental plaque biofilms. Bacterial viruses, also known as bacteriophages, have the capability of specifically infecting bacteria and effectively degrading bacterial biofilms. S. mutans phages, therefore, may prevent and control caries. Therapy based on phages has been applied in many fields, but the application of S. mutans phages in caries remains exploratory. This article will review the research progress of S. mutans phages in clinical caries prevention, aiming to provide a new idea for the clinical prevention of caries. The results of the literature review show that the living bacteriophage system has the advantages of high specificity, high affinity and good safety. However, due to its unstable structure, it can be processed into a more stable formulation by freeze-drying, spray drying, adding stability enhancers, or incorporating bacteriophages into ointments, biodegradable polymer matrices or particles to a certain extent to improve stability. The lysozyme produced by phages can digest the bacterial cell wall and release the assembled phage particles, which effectively cleave biofilms. In addition, the antigen binding fragment library for cariogenic pathogens was screened by phage display technology, and the purpose of caries prevention and treatment was achieved by passive immunization of antigen binding fragments. However, the host range of bacteriophages is narrow, so this kind of problem can be overcome by phage combined with traditional therapy or other drug use or cocktail therapy with multiple phages in clinical caries prevention and control.

The pathogenic mechanisms by which oral microbial communities affect the occurrence and development of dental caries remain unclear. Applications that make use of high⁃through put sequencing technologies and metaomics approaches to research dental caries etiology can provide new ideas about disease prevention and treatment. In this arti⁃ cle, we review advanced research presented in metaomics studies, including genomic, metagenomic, metatranscriptomic, metaproteomic and metabolomics studies of dental caries.

Dental caries is among the most prevalent chronic oral infectious diseases. Streptococcus mutans, a major cariogenic bacterial species, possesses several cariogenicity-associated characteristics, including exopolysaccharides (EPS) synthesis, biofilm formation, acidogenicity, and aciduricity. Nicotinamide (NAM), a form of vitamin B3, is a non-toxic, orally available, and inexpensive compound. The present study investigated the inhibitory effects of NAM on the cariogenic virulence factors of S. mutans in vitro and in vivo. NAM inhibited the growth of S. mutans UA159 and the clinical isolates. In addition, there was a decrease in the acid production and acid tolerance ability, as well as biofilm formation and EPS production of S. mutans after NAM treatment. Global gene expression profiling showed that 128 and 58 genes were significantly downregulated and upregulated, respectively, in NAM-treated S. mutans strains. The differentially expressed genes were mainly associated with carbohydrate transport and metabolism, glycolysis, acid tolerance. Moreover, in a rat caries model, NAM significantly reduced the incidence and severity of smooth and sulcal-surface caries in vivo. NAM exhibited good antimicrobial properties against S. mutans, indicating its potential value for antibiofilm and anti-caries applications. [ABSTRACT FROM AUTHOR]

Cyclic diadenosine monophosphate (c-di- AMP) has been implicated in the control of many important bacterial activities. However, the function of this molecule in S treptococcus mutans, the primary aetiological agent of human dental caries, is unknown. In this study, we identified and characterized a diadenylate cyclase, named CdaA, in S . mutans. Furthermore, we showed that in-frame deletion of the cdaA gene in S . mutans causes decreased c-di- AMP levels, increased sensitivity to hydrogen peroxide and increased production of extracellular polysaccharides. Global gene expression profiling revealed that more than 200 genes were significantly upregulated or downregulated (> 2.0-fold) in the cdaA mutant. Interestingly, genes with increased or decreased expression were clustered in cellular polysaccharide biosynthetic processes and oxidoreductase activity respectively. Notably, the expression of several genomic islands, such as GTF- B/ C, TnSmu, CRISPR1- Cas and CRISPR2- Cas, was found to be altered in the cdaA mutant, indicating a possible link between these genomic islands and c-di- AMP signalling. Collectively, the results reported here show that CdaA is an important global modulator in S . mutans and is required for optimal growth and environmental adaption. This report also paves the way to unveil further the roles of c-di- AMP signalling networks in the biology and pathogenicity of S . mutans. [ABSTRACT FROM AUTHOR]

Csn2 is an important protein of the CRISPR‐Cas system. The physiological function of this protein and its regulatory role in Streptococcus mutans, as the primary causative agent of human dental caries, is still unclear. In this study, we investigated whether csn2 deletion would affect S. mutans physiology and virulence gene expression. We used microscopic imaging, acid killing assays, pH drop, biofilm formation, and exopolysaccharide (EPS) production tests to determine whether csn2 deletion influenced S. mutans colony morphology, acid tolerance/production, and glucan formation abilities. Comparisons were made between quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) data from the UA159 and csn2 deletion strain to determine the impact of csn2 knockout on S. mutans gene expression. The results showed that deletion of S. mutanscsn2 changed its colony morphotype and made it more sensitive to acid. The expression levels of aciduricity genes, including leuA, leuB, leuC, and leuD, were significantly down‐regulated. Acid adaptation restored the aciduricity of csn2 mutant and enhanced the ability to synthesize EPS. The expression levels of EPS synthesis‐related genes, including gtfC and gtfD, were significantly up‐regulated after acid adaptation. In summary, deletion of S. mutanscsn2 exerted multiple effects on the virulence traits of this pathogen, including acid tolerance and EPS formation, and that these alterations could partially be attributed to changes in gene expression upon loss of csn2. Understanding the function of csn2 in S. mutans might lead to novel strategies to prevent or treat imbalances in oral microbiota that may favor diseases. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats with CRISPR-associated proteins (CRISPR-Cas) system, found in bacteria and archaea, provides sequence-based adaptive immunity against mobile genetic elements, including phages and plasmids. The oral cavity contains approximately 700 prokaryote species harboring known CRISPR-Cas systems, including type I, type II, type III, type V, and type VI, and unidentified CRISPR-Cas systems. There is increasing evidence to suggest that different CRISPR-Cas systems in the human oral microbiome can affect bacterial physiology through different mechanisms. Here, we review the canonical and novel functions of the CRISPR-Cas system, including defense against the invasion of foreign mobile elements, biofilm formation, acquisition of resistance genes, DNA repair, regulation of interspecific competition and intraspecific diversification, stress responses, and gene expression regulation. Overall, the mechanisms involved in CRISPR-Cas systems and their effects on bacterial physiology provide new insights into our understanding of the function and application of methods (including gene editing, modulation of CRISPR-Cas by anti-CRISPR, antimicrobials) on the oral microbiome. [ABSTRACT FROM AUTHOR]

Streptococcus mutans is the primary etiological agent of human dental caries. Its major virulence factors, glucosyltransferases (Gtfs), utilize sucrose to synthesize extracellular polysaccharides (EPS), leading to the formation of dental plaque biofilm. The current study was designed to develop a novel self-targeting gene editing technology that targeted gtfs to inhibit biofilms formation. The CRISPR-Cas system (ie, clustered regularly interspaced short palindromic repeat, with CRISPR-associated proteins) provides sequence-specific protection against foreign genetic materials in archaea and bacteria, and has been widely developed for genomic engineering. The first aim of this study was to test whether components of the CRISPR-Cas9 system from S mutans UA159 is necessary to defend against foreign DNA. The data showed that a suitable PAM site, tracrRNA, Cas9, and RNase III are indispensable elements to perform normal function of S mutans CRISPR-Cas9 system. Based on these results, we designed self-targeting CRISPR arrays (containing spacer sequences identifying with gtfB) and cloned them onto plasmids. Afterward, we transformed the plasmids and editing templates into UA159 (self-targeting) to acquire desired mutants. Our data showed that this technology performed well and was able to successfully edit gtfB or gtfBgtfC genes. This resulted in high reduction in EPS synthesis and was able to breakdown biofilm formation, which is also a promising tool for dental clinics in order to prevent the formation of S mutans biofilms in the future. [ABSTRACT FROM AUTHOR]