Oral microbiome sequencing revealed the enrichment of Fusobacterium sp., Porphyromonas sp., Campylobacter sp., and Neisseria sp. on the oral malignant fibroma surface of giant panda.

Introduction: Microbial community composition is closely associated with host disease onset and progression, underscoring the importance of understanding host-microbiota dynamics in various health contexts. Methods: In this study, we utilized full-length 16S rRNA gene sequencing to conduct species-level identification of the microorganisms in the oral cavity of a giant panda (Ailuropoda melanoleuca) with oral malignant fibroma. Results: We observed a significant difference between the microbial community of the tumor side and non-tumor side of the oral cavity of the giant panda, with the latter exhibiting higher microbial diversity. The tumor side was dominated by specific microorganisms, such as Fusobacterium simiae, Porphyromonas sp. feline oral taxon 110, Campylobacter sp. feline oral taxon 100, and Neisseria sp. feline oral taxon 078, that have been reported to be associated with tumorigenic processes and periodontal diseases in other organisms. According to the linear discriminant analysis effect size analysis, more than 9 distinct biomarkers were obtained between the tumor side and non-tumor side samples. Furthermore, the Kyoto Encyclopedia of Genes and Genomes analysis revealed that the oral microbiota of the giant panda was significantly associated with genetic information processing and metabolism, particularly cofactor and vitamin, amino acid, and carbohydrate metabolism. Furthermore, a significant bacterial invasion of epithelial cells was predicted in the tumor side. Discussion: This study provides crucial insights into the association between oral microbiota and oral tumors in giant pandas and offers potential biomarkers that may guide future health assessments and preventive strategies for captive and aging giant pandas.

[Characteristics of caries-related oral microorganisms in early childhood caries].

PURPOSE: To study the structural characteristics of oral microorganisms in children with caries by 16S rRNA high-throughput sequencing technology. METHODS: Thirty healthy children aged 3-5 years were enrolled as subjects. According to the index of dmfs, they were divided into caries-free (CF) group (15) and early childhood caries (ECC) group(15). To compare the differences in bacterial community structure, samples of saliva and dental plaque were collected, and high-throughput sequencing was conducted using the Illumina Miseq sequencing platform. Bioinformatics analysis was used to analyze the difference of microbial community structure and diversity with SPSS 23.0 software package. RESULTS: Microbial diversity in ECC group was significantly lower than CF group. At phylum level, Actinobateria was more abundant in saliva samples of ECC group, while Firmicutes was more abundant in plaque samples of CF group. At genus level, the abundance of Lautropia of CF group was higher in saliva samples while Cardiobacterium, Gemella and Granulicatella were abundant in plaque samples. The abundance of Rothia of ECC group was higher in saliva samples and Corynebacterium was abundant of ECC group in plaque samples. CONCLUSIONS: There are significant differences in the species and composition of microbial community in saliva and plaque of children with or without caries. Specific microorganisms are related to the occurrence of ECC, and screening specific microorganisms is helpful for early prediction and prevention of ECC.

Small RNA SmsR1 modulates acidogenicity and cariogenic virulence by affecting protein acetylation in Streptococcus mutans.

Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans, a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S. mutans, we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S. mutans. Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5'-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S. mutans, along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress.

SMU_1361c regulates the oxidative stress response of Streptococcus mutans.

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.IMPORTANCEStreptococcus 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.

Nicotinamide employs a starvation strategy against Porphyromonas gingivalis virulence by inhibiting the heme uptake system and gingipain activities.

Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B3 derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth-essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability.

Inhibition of Streptococcus mutans growth and biofilm formation through protein acetylation.

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.

FoxO1 Agonists Promote Bone Regeneration in Periodontitis by Protecting the Osteogenesis of Periodontal Ligament Stem Cells.

Protecting the function of periodontal ligament stem cells (PDLSCs) is crucial for bone regeneration in periodontitis. Forkhead box protein O1 (FoxO1) has been previously reported as a crucial mediator in bone homeostasis, providing a favorable environment for osteoblast proliferation and differentiation. In this study, we investigated the effect and mechanism of FoxO1 agonists on the osteogenesis of PDLSCs under inflammatory conditions. In this study, we screened FoxO1 agonists by detecting their effects on the osteogenic differentiation of PDLSCs. Then, the function of these agonists in bone regeneration was analyzed in the periodontitis model. We found that hyperoside or 2-furoyl-LIGRLO-amide trifluoroacetate salt (2-Fly) promoted osteogenic differentiation under inflammation by simultaneously inhibiting nuclear factor κB (NF-κB) activation, ß-catenin expression, and reactive oxygen species (ROS) production. Moreover, local injection of hyperoside or 2-Fly rescued the expression of FoxO1 and runt-related transcription factor 2 (Runx2) in vivo, alleviating alveolar bone loss and periodontal ligament damage. These findings suggested that FoxO1 agonists exerted a protective effect on osteogenesis in PDLSCs, as a result, facilitating bone formation under inflammatory conditions. Taken together, FoxO1 might serve as a therapeutic target for bone regeneration in periodontitis by mediating multiple signaling pathways.

vicR overexpression in Streptococcus mutans causes aggregation and affects interspecies competition.

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.

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.

Amelogenesis imperfecta in a Chinese family resulting from a FAM83H variation and the effect of FAM83H on the secretion of enamel matrix proteins.

OBJECTIVES: To investigate the variant of an amelogenesis imperfecta (AI) family and to explore the function of the FAM83H (family with sequence similarity 83 member H) in the enamel formation. MATERIALS AND METHODS: We investigated a five-generation Chinese family diagnosed with AI; clinical data was collected, whole-exome sequencing (WES) was conducted to explore the pathogenic gene and variants and Sanger sequencing was used to verify the variants. The three-dimensional protein structures of wild-type and mutant FAM83H were predicted using alpha fold 2. To study the possible regulatory function of Fam83h on amelogenesis, immunolocalization was performed to observe the expression of Fam83h protein in Sprague-Dawley rat postnatal incisors. The mRNA and protein level of amelogenin, enamelin, kallikrein-related peptidase-4 and ameloblastin were also detected after the Fam83h was knocked down by small interfering RNA (siRNA) in HAT-7 cells. RESULTS: A known nonsense variant (c.973 C > T) in exon 5 of FAM83H gene was found in this family, causing a truncated protein (p.R325X). Immunolocalization of Fam83h in Sprague-Dawley rat postnatal incisors showed that Fam83h protein expression was detected in presecretory and secretory stages. When Fam83h expression was reduced by siRNA, the expression of amelogenin, enamelin, kallikrein-related peptidase-4 decreased. However, the expression of ameloblastin increased. CONCLUSIONS: FAM83H gene variant (c.973 C > T) causes AI. FAM83H regulates the secretion of enamel matrix proteins and affects ameloblast differentiation. CLINICAL RELEVANCE: This study provided that FAM83H variants could influence enamel formation and provided new insights into the pathogenesis of AI.

Molecular and regulatory mechanisms of oxidative stress adaptation in Streptococcus mutans.

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.

Survival analysis of pulpectomy in primary molars performed under dental general anaesthesia: a two-year retrospective study.

BACKGROUND: To retrospectively investigate the success rate of primary-molar pulpectomy performed under general anaesthesia and the potential risk factors that affect the 24-month success rate. METHODS: The case data and two-year follow-up records of children (aged 3-6 years) who received pulpectomy in primary molars performed under general anaesthesia were reviewed and assessed. Potential risk factors included age, gender, decayed-missing-filled teeth, endodontic diagnosis, tooth location, and postobturation sealing of the pulp chamber floor with MTA. With a two-year follow-up period, the outcomes of all the primary molars were classified into success and failure. Survival analysis was used to assess the outcomes. The Kaplan-Meier method was used to analyse the success rate. Univariate and multivariate Cox proportional hazards regression models were used to evaluate the potential risk factors associated with the overall survival of primary molars. RESULTS: A total of 410 teeth from 163 children (88 boys and 75 girls) were included in this study. The overall two-year success rate was 66.1% for all primary molars. The mean overall survival time for this study was 22.1 (95% CI, 21.73‒22.48) months. Multivariate Cox regression analysis demonstrated that endodontic diagnosis (irreversible pulpitis or periapical periodontitis), tooth location (maxillary or mandibular primary molar), and postobturation sealing of the pulp chamber floor (MTA or no-MTA) were significant risk factors for overall survival in this study (P < .05). The differences in success rates were not statistically significant in terms of age, gender, and decayed-missing-filled teeth (P > .05). CONCLUSIONS: When compared to teeth diagnosed with irreversible pulpitis, those with periapical periodontitis failed more frequently. Postobturation sealing of the pulp chamber floor with MTA improved the success rate of pulpectomy in primary molars, especially when the inflammation did not spread to the periradicular area.

Candida albicans CHK1 gene regulates its cross-kingdom interactions with Streptococcus mutans to promote caries.

The cross-kingdom interactions between Candida albicans and Streptococcus mutans have played important roles in early childhood caries (ECC). However, the key pathways of C. albicans promoting the cariogenicity of S. mutans are still unclear. Here, we found that C. albicans CHK1 gene was highly upregulated in their dual-species biofilms. C. albicans chk1Δ/Δ significantly reduced the synergistical growth promotion, biofilm formation, and exopolysaccharides (EPS) production of S. mutans, the key cariogenic agent, compared to C. albicans wild type (WT) and CHK1 complementary strains. C. albicans WT upregulated the expressions of S. mutans EPS biosynthesis genes gtfB, gtfC, and gtfD, and their regulatory genes vicR and vicK, but chk1Δ/Δ had no effects. Both C. albicans WT and chk1Δ/Δ failed to promote the biofilm formation and EPS production of S. mutans ΔvicK and antisense-vicR strains, indicating that C. albicans CHK1 upregulated S. mutans vicR and vicK to increase the EPS biosynthesis gene expression, then enhanced the EPS production and biofilm formation to promote the cariogenicity. In rat caries model, the coinfection with chk1Δ/Δ and S. mutans decreased the colonization of S. mutans and developed less caries especially the severe caries compared to that from the combinations of S. mutans with C. albicans WT, indicating the essential role of C. albicans CHK1 gene in the development of dental caries. Our study for the first time demonstrated the key roles of C. albicans CHK1 gene in dental caries and suggested that it may be a practical target to reduce or treat ECC. KEY POINTS: • C. albicans CHK1 gene is important for its interaction with S. mutans. • CHK1 regulates S. mutans two-component system to promote its cariogenicity. • CHK1 gene regulates the cariogenicity of S. mutans in rat dental caries.

Acetylation of Lactate Dehydrogenase Negatively Regulates the Acidogenicity of Streptococcus mutans.

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.

AhrC Negatively Regulates Streptococcus mutans Arginine Biosynthesis.

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 ß-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.

Nicotinamide could reduce growth and cariogenic virulence of Streptococcus mutans.

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.

Post-translational Modifications in Oral Bacteria and Their Functional Impact.

Oral bacteria colonize the oral cavity, surrounding complex and variable environments. Post-translational modifications (PTMs) are an efficient biochemical mechanism across all domains of life. Oral bacteria could depend on PTMs to quickly regulate their metabolic processes in the face of external stimuli. In recent years, thanks to advances in enrichment strategies, the number and variety of PTMs that have been identified and characterized in oral bacteria have increased. PTMs, covalently modified by diverse enzymes, occur in amino acid residues of the target substrate, altering the functions of proteins involved in different biological processes. For example, Ptk1 reciprocally phosphorylates Php1 on tyrosine residues 159 and 161, required for Porphyromonas gingivalis EPS production and community development with the antecedent oral biofilm constituent Streptococcus gordonii, and in turn Php1 dephosphorylates Ptk1 and rapidly causes the conversion of Ptk1 to a state of low tyrosine phosphorylation. Protein acetylation is also widespread in oral bacteria. In the acetylome of Streptococcus mutans, 973 acetylation sites were identified in 445 proteins, accounting for 22.7% of overall proteins involving virulence factors and pathogenic processes. Other PTMs in oral bacteria include serine or threonine glycosylation in Cnm involving intracerebral hemorrhage, arginine citrullination in peptidylarginine deiminases (PADs), leading to inflammation, lysine succinylation in P. gingivalis virulence factors (gingipains, fimbriae, RagB, and PorR), and cysteine glutathionylation in thioredoxin-like protein (Tlp) in response to oxidative stress in S. mutans. Here we review oral bacterial PTMs, focusing on acetylation, phosphorylation, glycosylation, citrullination, succinylation, and glutathionylation, and corresponding modifying enzymes. We describe different PTMs in association with some examples, discussing their potential role and function in oral bacteria physiological processes and regulatory networks. Identification and characterization of PTMs not only contribute to understanding their role in oral bacterial virulence, adaption, and resistance but will open new avenues to treat oral infectious diseases.

Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence.

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.

Molecular mechanisms of inhibiting glucosyltransferases for biofilm formation in Streptococcus mutans.

Glucosyltransferases (Gtfs) play critical roles in the etiology and pathogenesis of Streptococcus mutans (S. mutans)- mediated dental caries including early childhood caries. Gtfs enhance the biofilm formation and promotes colonization of cariogenic bacteria by generating biofilm extracellular polysaccharides (EPSs), the key virulence property in the cariogenic process. Therefore, Gtfs have become an appealing target for effective therapeutic interventions that inhibit cariogenic biofilms. Importantly, targeting Gtfs selectively impairs the S. mutans virulence without affecting S. mutans existence or the existence of other species in the oral cavity. Over the past decade, numerous Gtfs inhibitory molecules have been identified, mainly including natural and synthetic compounds and their derivatives, antibodies, and metal ions. These therapeutic agents exert their inhibitory role in inhibiting the expression gtf genes and the activities and secretion of Gtfs enzymes with a wide range of sensitivity and effectiveness. Understanding molecular mechanisms of inhibiting Gtfs will contribute to instructing drug combination strategies, which is more effective for inhibiting Gtfs than one drug or class of drugs. This review highlights our current understanding of Gtfs activities and their potential utility, and discusses challenges and opportunities for future exploration of Gtfs as a therapeutic target.

Transcriptional Profiling Reveals the Importance of RcrR in the Regulation of Multiple Sugar Transportation and Biofilm Formation in Streptococcus mutans.

The ability of Streptococcus mutans to survive and cause dental caries is dependent on its ability to metabolize various carbohydrates, accompanied by extracellular polysaccharide synthesis and biofilm formation. Here, the role of an rel competence-related regulator (RcrR) in the regulation of multiple sugar transportation and biofilm formation is reported. The deletion of the rcrR gene in S. mutans caused delayed growth, decreased biofilm formation ability, and affected the expression level of its multiple sugar transportation-related genes. Transcriptional profiling revealed 17 differentially expressed genes in the rcrR mutant. Five were downregulated and clustered with the sugar phosphotransferase (PTS) systems (mannitol- and trehalose-specific PTS systems). The conserved sites bound by the rcrR promoter were then determined by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting assays. Furthermore, a potential binding motif in the promoters of the two PTS operons was predicted using MEME Suite 5.1.1. RcrR could bind to the promoter regions of the two operons in vitro, and the sugar transporter-related genes of the two operons were upregulated in an rcrR-overexpressing strain. In addition, when RcrR-binding sites were deleted, the growth rates and final yield of S. mutans were significantly decreased in tryptone-vitamin (TV) medium supplemented with different sugars, but not in absolute TV medium. These results revealed that RcrR acted as a transcription activator to regulate the two PTS systems, accompanied by multiple sugar transportation and biofilm formation. Collectively, these results indicate that RcrR is a critical transcription factor in S. mutans that regulates bacterial growth, biofilm formation, and multiple sugar transportation. IMPORTANCE The human oral cavity is a constantly changing environment. Tooth decay is a commonly prevalent chronic disease mainly caused by the cariogenic bacterium Streptococcus mutans. S. mutans is an oral pathogen that metabolizes various carbohydrates into extracellular polysaccharides (EPSs), biofilm, and tooth-destroying lactic acid. The host diet strongly influences the availability of multiple carbohydrates. Here, we showed that the RcrR transcription regulator plays a significant role in the regulation of biofilm formation and multiple sugar transportation. Further systematic evaluation of how RcrR regulates the transportation of various sugars and biofilm formation was also conducted. Notably, this study decrypts the physiological functions of RcrR as a potential target for the better prevention of dental caries.