The Regulator Gene rnc Is Closely Involved in Biofilm Formation in Streptococcus mutans.

Streptococcus mutans is an important factor in the etiology and pathogenesis of dental caries, largely owing to its ability to form a stable biofilm. Previous animal studies have indicated that rnc could decrease the amount of sulcal caries, and that the downregulation of cariogenicity might be due to its capacity to disrupt biofilm formation. However, the biofunctions by which rnc is involved in biofilm formation remain to be elucidated. In this study, we further investigate the role of rnc based on the study of mature biofilm. Scanning electron microscopy and the crystal violet assay were used to detect the biofilm forming ability. The production and distribution of exopolysaccharides within biofilm was analyzed by exopolysaccharide staining. Gel permeation chromatography was used to perform molecular weight assessment. Its adhesion force was measured by atomic force microscopy. The expression of biofilm formation-associated genes was analyzed at the mRNA level by qPCR. Here, we found that rnc could occur and function in biofilm formation by assembling well-structured, exopolysaccharide-encased, stable biofilms in S. mutans. The weakened biofilm forming ability of rnc-deficient strains was associated with the reduction of exopolysaccharide production and bacterial adhesion. Over all, these data illustrate an interesting situation in which an unappreciated regulatory gene acquired for virulence, rnc, most likely has been coopted as a potential regulator of biofilm formation in S. mutans. Further characterization of rnc may lead to the identification of a possible pathogenic biofilm-specific treatment for dental caries.

Role of connexin 43 in odontoblastic differentiation and structural maintenance in pulp damage repair.

Dental pulp can initiate its damage repair after an injury of the pulp-dentin complex by rearrangement of odontoblasts and formation of newly differentiated odontoblast-like cells. Connexin 43 (Cx43) is one of the gap junction proteins that participates in multiple tissue repair processes. However, the role of Cx43 in the repair of the dental pulp remains unclear. This study aimed to determine the function of Cx43 in the odontoblast arrangement patterns and odontoblastic differentiation. Human teeth for in vitro experiments were acquired, and a pulp injury model in Sprague-Dawley rats was used for in vivo analysis. The odontoblast arrangement pattern and the expression of Cx43 and dentin sialophosphoprotein (DSPP) were assessed. To investigate the function of Cx43 in odontoblastic differentiation, we overexpressed or inhibited Cx43. The results indicated that polarized odontoblasts were arranged along the pulp-dentin interface and had high levels of Cx43 expression in the healthy teeth; however, the odontoblast arrangement pattern was slightly changed concomitant to an increase in the Cx43 expression in the carious teeth. Regularly arranged odontoblast-like cells had high levels of the Cx43 expression during the formation of mature dentin, but the odontoblast-like cells were not regularly arranged beneath immature osteodentin in the pulp injury models. Subsequent in vitro experiments demonstrated that Cx43 is upregulated during odontoblastic differentiation of the dental pulp cells, and inhibition or overexpression of Cx43 influence the odontoblastic differentiation. Thus, Cx43 may be involved in the maintenance of odontoblast arrangement patterns, and influence the pulp repair outcomes by the regulation of odontoblastic differentiation.

Regulation of water-soluble glucan synthesis by the Streptococcus mutans dexA gene effects biofilm aggregation and cariogenic pathogenicity.

The cariogenic pathogen Streptococcus mutans effectively utilizes dietary sucrose for the synthesis of exopolysaccharides (EPS), which act as a scaffold for its biofilm and thus contribute to its cariogenic pathogenicity. Dextranase (Dex), which is a type of glucanase, participates in the degradation of water-soluble glucan (WSG); however, the structural features of the EPS regulated by the dexAgene have received limited attention. Our recent studies reported novel protocols to fractionate and analyzed the structural characteristics of glucans from S mutans biofilms. In this study, we identify the role of the S mutans dexAgene in dextran-dependent aggregation in biofilm formation. Our results show that deletion of dexA (SmudexA) results in increased transcription of EPS synthesis-related genes, including gtfB, gtfD, and ftf. Interestingly, we reveal that inactivating the dexA gene may lead to elevated WSG synthesis in S mutans , which results in dysregulated cariogenicity in vivo. Furthermore, structural analysis provides new insights regarding the lack of mannose monosaccharides, especially in the WSG synthesis of the SmudexA mutants. The biofilm phenotypes that are associated with the reduced glucose monosaccharide composition in both WSG and water-insoluble glucan shift the dental biofilm to reduce the cariogenic incidence of the SmudexA mutants. Taken together, these data reveal that EPS synthesis fine-tuning by the dexA gene results in a densely packed EPS matrix that may impede the glucose metabolism of WSG, thereby leading to the lack of an energy source for the bacteria. These results highlight dexA targeting as a potentially effective tool in dental caries management.

The rnc Gene Promotes Exopolysaccharide Synthesis and Represses the vicRKX Gene Expressions via MicroRNA-Size Small RNAs in Streptococcus mutans.

Dental caries is a biofilm-dependent disease that largely relies on the ability of Streptococcus mutans to synthesize exopolysaccharides. Although the rnc gene is suggested to be involved in virulence mechanisms in many other bacteria, the information regarding it in S. mutans is very limited. Here, using deletion or overexpression mutant assay, we demonstrated that rnc in S. mutans significantly positively regulated exopolysaccharide synthesis and further altered biofilm formation. Meanwhile, the cariogenecity of S. mutans was decreased by deletion of rnc in a specific pathogen-free (SPF) rat model. Interestingly, analyzing the expression at mRNA level, we found the downstream vic locus was repressed by rnc in S. mutans. Using deep sequencing and bioinformatics analysis, for the first time, three putative microRNA-size small RNAs (msRNAs) targeting vicRKX were predicted in S. mutans. The expression levels of these msRNAs were negatively correlated with vicRKX but positively correlated with rnc, indicating rnc probably repressed vicRKX expression through msRNAs at the post-transcriptional level. In all, the results present that rnc has a potential role in the regulation of exopolysaccharide synthesis and can affect vicRKX expressions via post-transcriptional repression in S. mutans. This study provides an alternative avenue for further research aimed at preventing caries.

Modulation of Biofilm Exopolysaccharides by the Streptococcus mutans vicX Gene.

The cariogenic pathogen Streptococcus mutans effectively utilizes dietary sucrose for the synthesis of exopolysaccharide, which act as a scaffold for its biofilm, thus contributing to its pathogenicity, environmental stress tolerance, and antimicrobial resistance. The two-component system VicRK of S. mutans regulates a group of virulence genes that are associated with biofilm matrix synthesis. Knockout of vicX affects biofilm formation, oxidative stress tolerance, and transformation of S. mutans. However, little is known regarding the vicX-modulated structural characteristics of the exopolysaccharides underlying the biofilm formation and the phenotypes of the vicX mutants. Here, we identified the role of vicX in the structural characteristics of the exopolysaccharide matrix and biofilm physiology. The vicX mutant (SmuvicX) biofilms seemingly exhibited "desertification" with architecturally impaired exopolysaccharide-enmeshed cell clusters, compared with the UA159 strain (S. mutans wild type strain). Concomitantly, SmuvicX showed a decrease in water-insoluble glucan (WIG) synthesis and in WIG/water-soluble glucan (WSG) ratio. Gel permeation chromatography (GPC) showed that the WIG isolated from the SmuvicX biofilms had a much lower molecular weight compared with the UA159 strain indicating differences in polysaccharide chain lengths. A monosaccharide composition analysis demonstrated the importance of the vicX gene in the glucose metabolism. We performed metabolite profiling via (1)H nuclear magnetic resonance spectroscopy, which showed that several chemical shifts were absent in both WSG and WIG of SmuvicX biofilms compared with the UA159 strain. Thus, the modulation of structural characteristics of exopolysaccharide by vicX provides new insights into the interaction between the exopolysaccharide structure, gene functions, and cariogenicity. Our results suggest that vicX gene modulates the structural characteristics of exopolysaccharide associated with cariogenicity, which may be explored as a potential target that contributes to dental caries management. Furthermore, the methods used to purify the EPS of S. mutans biofilms and to analyze multiple aspects of its structure (GPC, gas chromatography-mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy) may be useful approaches to determine the roles of other virulence genes for dental caries prevention.