The LiaSR Two-Component System Regulates Resistance to Chlorhexidine in Streptococcus mutans.

Chlorhexidine (CHX) is widely considered to be the gold standard for preventing dental caries. However, it is possible to induce resistance to CHX. The LiaSR two-component system has been identified that contributed to CHX resistance in Streptococcus mutans, which is one of the major pathogens in dental caries. However, the underlying mechanisms remain unclear. In this study, an MIC assay and a viability assessment demonstrated that after deleting the liaS and liaR genes, the sensitivity of mutants could increase. The Nile Red efflux assay exhibited that the efflux rates of mutants were significantly decreased. The RT-qPCR results indicated that the LiaSR two-component system-mediating influence on the expression of lmrB in S. mutans contributed to the efflux rate. The hydrophobicity assay and membrane potential assay showed that the mutants had higher levels of hydrophobicity and depolarization, suggesting that their membranes were more easily disturbed. The TEM graphs revealed that the border of the cell membrane was unclear in mutants compared with the wild-type strain, indicating that the cell envelope's stress response may have been inhibited. While the surface charge of mutants showed no significant difference in the wild-type strain according to the result of cytochrome c-based charged determination. This study provides valuable novel insights into the mechanisms of the LiaSR two-component system in the CHX resistance of S. mutans.

Current and prospective therapeutic strategies: tackling Candida albicans and Streptococcus mutans cross-kingdom biofilm.

Candida albicans (C. albicans) is the most frequent strain associated with cross-kingdom infections in the oral cavity. Clinical evidence shows the co-existence of Streptococcus mutans (S. mutans) and C. albicans in the carious lesions especially in children with early childhood caries (ECC) and demonstrates the close interaction between them. During the interaction, both S. mutans and C. albicans have evolved a complex network of regulatory mechanisms to boost cariogenic virulence and modulate tolerance upon stress changes in the external environment. The intricate relationship and unpredictable consequences pose great therapeutic challenges in clinics, which indicate the demand for de novo emergence of potential antimicrobial therapy with multi-targets or combinatorial therapies. In this article, we present an overview of the clinical significance, and cooperative network of the cross-kingdom interaction between S. mutans and C. albicans. Furthermore, we also summarize the current strategies for targeting cross-kingdom biofilm.

Dlt operon regulates physiological function and cariogenic virulence in Streptococcus mutans.

Streptococcus mutans is one of the major cariogenic pathogens in the oral cavity. The dlt operon is responsible for the process of D-alanylation of lipoteichoic acid and is related to the virulence of S. mutans. The dlt operon contributes to the adhesion, biofilm formation, stress response, interspecies competitiveness and autolysis of S. mutans. In addition, we have summarized the possible regulatory networks of the dlt operon. This review highlights the significant role of the dlt operon in S. mutans and provides new ideas for ecological caries prevention.

What is this summary about? Dental caries is a common oral disease that destroys teeth. Streptococcus mutans is the major pathogen of dental caries and its cariogenic virulence factors depend on the ability of biofilm formation, acid production and tolerance, stress response, and interspecific competitiveness. The dlt operon is responsible for the process of D-alanylation of lipoteichoic acid and is related to the virulence of Gram-positive bacteria. To understand the role of the dlt operon in S. mutans, this review summarized this based on previous studies. What were the results? The dlt operon can regulate the D-alanylation of lipoteichoic acid to affect the cell surface charge of S. mutans. The change in characteristics of the cell wall will affect the physiological functions of S. mutans, such as adhesion, biofilm formation, stress response, autolysis and interspecies competitiveness. However, the regulatory mechanism of the dlt operon is still unclear and needs further study. What do the results of the study mean? This review highlights the significant role of the dlt operon in S. mutans, which will increase the understanding of the cariogenic mechanism of S. mutans and provide new ideas for ecological caries prevention, such as the development of antibacterial agents targeting the dlt operon.

D-alanylation of lipoteichoic acid contributes to biofilm formation and acidogenesis capacity of Streptococcusmutans.

BACKGROUND: D-alanylation of Lipoteichoic acid (LTA) is considered to be essential for virulence factors expression in Gram-positive microorganism. The effects of the D-alanylation of LTA on biofilm formation and acidogenesis of Streptococcus mutans (S. mutans) are still not clearly understood. AIM: This study was designed to investigate the impact of D-alanylation of LTA on biofilm formation and acidogenesis of S. mutans and explore the related mechanisms. METHODS AND MATERIAL: We compared the biofilm formation process by fluorescence microscope observation of LTA D-alanylation blocking strain with that of the wildtype strain. Auto-aggregation, cell surface charge, and polysaccharide production assays were performed to investigate the related mechanisms. pH drop assay and glycolysis pH drop-down analysis were carried out to evaluate the acidogenesis capacity of S. mutans after LTA D-alanylation blocking. To identify the biofilm formation and adhesive-related genes expressions of S. mutans mutant, qRT-PCR was performed. RESULTS: After blocking off the D-alanylation of LTA, S. mutans could not form the three-dimensional structural biofilm, in which cells were scattered on the substratum as small clusters. The auto-aggregation was prompted due to the mutant strain cell morphology change (*p < 0.05). Furthermore, more negative charges were found on the mutant strain cells surfaces and fewer water-insoluble glucans were produced in mutant biofilm (*p < 0.05). The adhesion capacity of the S. mutans biofilm was impaired after LTA D-alanylation blocking (*p < 0.05). Biofilm formation and adhesive-related genes expressions decreased (*p < 0.05), especially at the early stages of biofilm formation. S. mutans mutant strains exhibited suppressed acidogenesis because its glycolytic activity was impaired. CONCLUSION: The results of this study suggest that blocking of LTA D-alanylation disrupts normal biofilm formation in S. mutans predominantly if not entirely by altering intercellular auto-aggregation, cell adhesion, and extracellular matrix formation. Moreover, our study results suggest that the LTA D-alanylation plays an important role in S. mutans acidogenesis by altering glycolytic activity. These findings add to the knowledge about mechanisms underlying biofilm formation and acid tolerance in S. mutans.

[Effect of Lipoteichoic Acid Synthesis-Related Gene dltD on Acid Tolerance of Highly Cariogenic Strains of Streptococcus mutans].

Objective: To study the role and possible mechanism of dltD in the acid tolerance of Streptococcus mutans 593 (SM593), and to provide a theoretical basis for the ecological prevention and control of dental caries by constructing the dltD gene deletion strain of SM593 (SM593-ΔdltD). Methods: 1) SM593-Δ dltD was constructed by homologous recombination. 2) The growth curve of SM593 dltD and SM593-Δ dltD under different pH culture conditions was drawn by the automatic growth curve analyzer to compare their acid tolerance. Colony forming unit (CFU) at different time points was used to calculate the survival rate and to compare the acid tolerance response (ATR) of SM593 and SM593-Δ dltD. 3) Under different pH conditions, glycolysis experiments, proton permeability test and H +-ATPase activity test were conducted to make preliminary exploration into the mechanisms of how dltD gene deletion may affect acid tolerance. Results: 1) PCR and sequencing results showed that the SM593-Δ dltD was constructed successfully. 2) With decreasing pH value of the culture medium, the growth of SM593-Δ dltD slowed down. When the pH value of the culture medium was 5.0, SM593-Δ dltD was not allowed to grow, and its acid tolerance was lower than that of SM593. Compared with SM593, the ATR capability of SM593-Δ dltD was decreased. 3) SM593 dltD and SM593-Δ dltD did not show obvious difference in their glycolysis ability under different pH conditions. Compared with SM593 dltD, the proton permeability of SM593-Δ dltD under different pH conditions was increased significantly (P<0.05), and H +-ATPase activity decreased significantly (P<0.05). Conclusion: Compared with SM593 dltD, SM593-Δ dltD showed obvious decrease in acid tolerance, which may be caused by the significant increase in proton permeability and significant decrease in the H +-ATPase activity induced by the deletion of the dltD gene, hence reducing its ability to maintain intracellular pH homeostasis.

The dlt operon contributes to the resistance to chlorhexidine in Streptococcus mutans.

Chlorhexidine (CHX) is considered to be the gold standard for dental caries prevention and is widely applied in dental practice. However, the long-term application of CHX may result in CHX-resistance in oral pathogens. The aim of this study was to investigate whether long-term use of CHX causes resistance in Streptococcus mutans and to explore the possible associated mechanisms. Four different S. mutans strains were chosen for this study to exclude the specificity of strains. The four strains displayed an increase in minimum inhibitory concentration (MIC) after exposure to CHX for 10 passages. The features and cariogenicity of S. mutans CHX-resistant strains (SM-Cs) that were exposed to CHX for 10 passages with increased MIC did not differ significantly to the parental strains. The SM-Cs were more hydrophobic than the parental strains. The dltC and dltD genes were upregulated in SM-Cs. Relative expression of the BceA, BceR, and SMU.862 genes in SM-Cs was similar to or lower than that of the parental strains. The MIC value was significantly lower in dltC knockout mutants. These findings confirmed that continuous exposure to CHX could induce CHX-resistance in S. mutans. The increased cell surface hydrophobicity and upregulated expression of dlt operon were possible underlying mechanisms of CHX-resistance in S. mutans.

Role of D-alanylation of Streptococcus mutans lipoteichoic acid in interspecies competitiveness.

BACKGROUND: The D-alanylation of lipoteichoic acid (LTA) is essential for the physiological metabolism of Streptococcus mutans (S. mutans). This study was designed to investigate the influence of D-alanylation of LTA on interspecies competitiveness of S. mutans. METHODS: The process of D-alanylation was blocked by the inactivation of dltC. Agar competition assays, conditioned medium assays, and qRT-PCR were used to evaluate the production of antimicrobial compounds in S. mutans mutant. Dual-species biofilm was formed to investigate the competitiveness of S. mutans mutant cocultured with S. sanguinis or S. gordonii. RESULTS: S. mutans mutant could not produce antimicrobial compounds efficiently when cocultured with commensal bacteria (*p < 0.05). The mutant showed compromised competitiveness in dual-species biofilms. The ratio of the mutant in dual-species biofilms decreased, and the terminal pH of the culture medium in mutant groups (mutant+S. sanguinis/S. gordonii) was higher than that in wild-type groups (*p < 0.05). Scanning electron microscope (SEM) showed weaker demineralization of enamel treated with dual-species biofilms consisting of mutant and commensal bacteria. CONCLUSION: D-Alanylation is involved in interspecies competitiveness of S. mutans within oral biofilm by regulating mutacins and lactic acid production, which may modulate the profiles of dental biofilms. Results provide new insights into dental caries prevention and treatment.

Disinfection of Cariogenic Pathogens in Planktonic Lifestyle, Biofilm and Carious Dentine with Antimicrobial Photodynamic Therapy.

Antimicrobial photodynamic therapy (aPDT) has been recommended for clinical application. Its antibacterial effect on bacteria remained in dentinal tubule was seldom investigated. Here, we evaluated the antibacterial effects of aPDT on Streptococcus mutans (S. mutans) and Lactobacillus acidophilus (L. acidophilus) in planktonic lifestyle, biofilm and carious dentine. Mono-species biofilms or dentinal caries formed on human dentine slices or slabs. Bacterial suspension, biofilms and dentine caries were treated with 0.1 mg mL-1 toluidine Blue O followed by irradiation with a light emission diode (λ - 635 ± 10 nm; 500 mW; 31.5 J cm-2 ; 60 s) and 0.12% chlorhexidine (CHX), respectively. Residual bacteria were determined by microbial culture analysis and scanning electron microscopy (SEM). One-way analysis of variance (ANOVA) was performed to detect the significance of the variables. Both treatments significantly reduced the number of L. acidophilus in planktonic state, biofilm and carious dentine (P < 0.05). For S. mutans, CHX was only bactericidal against suspension (P < 0.05), while aPDT was effective on both suspension and biofilm (P < 0.05) while not for dentin caries (P > 0.05). Under the experimental conditions assessed, aPDT could be an alternative disinfection method for superficial layer of caries cavity. Its disinfection on bacteria in dentinal tubule of deep layer was deficient.