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Streptococcus mutans constantly coexists with Candida albicans in plaque biofilms of early childhood caries (ECC). The progression of ECC can be influenced by the interactions between S. mutans and C. albicans through exopolysaccharides (EPS). Our previous studies have shown that rnc, the gene encoding ribonuclease III (RNase III), is implicated in the cariogenicity of S. mutans by regulating EPS metabolism. The DCR1 gene in C. albicans encodes the sole functional RNase III and is capable of producing non-coding RNAs. However, whether rnc or DCR1 can regulate the structure or cariogenic virulence of the cross-kingdom biofilm of S. mutans and C. albicans is not yet well understood. By using gene disruption or overexpression assays, this study aims to investigate the roles of rnc and DCR1 in modulating the biological characteristics of dual-species biofilms of S. mutans and C. albicans and to reveal the molecular mechanism of regulation. The morphology, biomass, EPS content, and lactic acid production of the dual-species biofilm were assessed. Quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptomic profiling were performed to unravel the alteration of C. albicans virulence. We found that both rnc and DCR1 could regulate the biological traits of cross-kingdom biofilms. The rnc gene prominently contributed to the formation of dual-species biofilms by positively modulating the extracellular polysaccharide synthesis, leading to increased biomass, biofilm roughness, and acid production. Changes in the microecological system probably impacted the virulence as well as polysaccharide or pyruvate metabolism pathways of C. albicans, which facilitated the assembly of a cariogenic cross-kingdom biofilm and the generation of an augmented acidic milieu. These results may provide an avenue for exploring new targets for the effective prevention and treatment of ECC. [ABSTRACT FROM AUTHOR]

Streptococcus mutans is known as the crucial pathogen of human dental caries, owing to its contribution to the biofilm development via the capacity of synthesizing exopolysaccharide (EPS), which mainly compose of α-glycosidic bond and β-glycosidic bond. β-glycosidic bond is less flexible than α-glycosidic bond because of differences between their configurational properties. Previous studies have shown that the rnc gene is implicated in the EPS formation and the cariogenicity of S. mutans. However, the effects of rnc on the microstructure of EPS have been not well-understood yet. Here, we further investigated how the rnc gene worked to modulate microstructural properties of the extracellular polysaccharide of S. mutans using glycomics methods. The gas chromatography-mass spectrometer showed that the proportion of glucose was decreased in water-soluble EPS and galactose was absent in water-insoluble EPS from the S. mutans rnc-deficient strain (Smurnc), compared with the isogenic wild-type strain (UA159). The composition of functional groups and the displacement of hydrogen bond were analyzed by infrared radiation and 1H nuclear magnetic resonance, respectively. In addition, phenotypic modulation of the biofilm matrix was assessed by microscopy. We found that the EPS of UA159 and the rnc overexpression strain (Smurnc+) mainly consisted of β-glycosidic bonds. Conversely, the EPS of Smurnc were made up of mostly α-glycosidic bonds, leading to the attenuation of biofilm biomass and bacterial adhesion. Furthermore, the existence of β-glycosidic bond was verified by enzyme digestion. Collectively, the rnc gene modulates the conversion of β-glycosidic bonds, which may play important roles in regulating the micromolecule structure of the EPS matrix, thus affecting the characteristics of S. mutans biofilm. These data illustrate that β-glycosidic bonds mediated by rnc may be potential targets for the prevention and treatment of dental caries. [ABSTRACT FROM AUTHOR]

Objective To study the changes of acid resistance, oxidation resistance and high osmotic pressure resis⁃ tance of Streptococcus mutans after knockout of rnc gene and its possible regulatory mechanism. Methods Through PCR ligation mutagenesis, an rnc knockout mutant (Smurnc) was constructed. Acid tolerance, oxidation tolerance and high osmotic pressure tolerance were compared between Smurnc and the wild strain respectively. Real⁃time RT⁃PCR was used to verify the changes in expression of stress tolerance related genes at the transcriptional level. Results When rnc gene was knocked out, the acid tolerance (c2=13.464, P=0.001) and oxidation tolerance (c2=4.505, P=0.048) of Streptococcus mutans was significantly decreased, but the high osmotic pressure tolerance was significantly increased (c2=11.971, P=0.001). Expression of stress tolerance related genes luxS and ropA (0.64 and 0.51 times expression of the wild strain) had been significantly downregulated (P

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. [ABSTRACT FROM AUTHOR]

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, we demonstrated that deletion or overexpression of 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. We used deep sequencing and bioinformatics analysis to predict, for the first time, three putative microRNA-size small RNAs (msRNAs) targeting vicRKX 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.