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.

Regulatory mechanisms of exopolysaccharide synthesis and biofilm formation in Streptococcus mutans.

Background: Dental caries is a chronic, multifactorial and biofilm-mediated oral bacterial infection affecting almost every age group and every geographical region. Streptococcus mutans is considered an important pathogen responsible for the initiation and development of dental caries. It produces exopolysaccharides in situ to promote the colonization of cariogenic bacteria and coordinate dental biofilm development. Objective: The understanding of the regulatory mechanism of S. mutans biofilm formation can provide a theoretical basis for the prevention and treatment of caries. Design: At present, an increasing number of studies have identified many regulatory systems in S. mutans that regulate biofilm formation, including second messengers (e.g. c-di-AMP, Ap4A), transcription factors (e.g. EpsR, RcrR, StsR, AhrC, FruR), two-component systems (e.g. CovR, VicR), small RNA (including sRNA0426, srn92532, and srn133489), acetylation modifications (e.g. ActG), CRISPR-associated proteins (e.g. Cas3), PTS systems (e.g. EIIAB), quorum-sensing signaling system (e.g. LuxS), enzymes (including Dex, YidC, CopZ, EzrA, lmrB, SprV, RecA, PdxR, MurI) and small-molecule metabolites. Results: This review summarizes the recent progress in the molecular regulatory mechanisms of exopolysaccharides synthesis and biofilm formation in S. mutans.

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.

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.

Deletion of the yqeK gene leads to the accumulation of Ap4A and reduced biofilm formation in Streptococcus mutans.

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.