Supplementary Material for: The Regulator Gene <b><i>rnc</i></b> Is Closely Involved in Biofilm Formation in <b><i>Streptococcus mutans</i></b>

<i>Streptococcus mutans</i> 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 <i>rnc</i> 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 <i>rnc</i> is involved in biofilm formation remain to be elucidated. In this study, we further investigate the role of <i>rnc</i> 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 <i>rnc</i> could occur and function in biofilm formation by assembling well-structured, exopolysaccharide-encased, stable biofilms in <i>S. mutans</i>. The weakened biofilm forming ability of <i>rnc</i>-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, <i>rnc</i>, most likely has been coopted as a potential regulator of biofilm formation in <i>S. mutans</i>. Further characterization of <i>rnc</i> may lead to the identification of a possible pathogenic biofilm-specific treatment for dental caries.

变形链球菌（Streptococcus mutans）是龋齿（dental caries）病因与发病机制的重要致病因素，这主要源于其可形成稳定生物被膜（biofilm）的能力。既往动物实验表明，rnc可减少窝沟龋齿的发生量，其致龋性下调可能与其破坏生物被膜形成的能力相关。然而，rnc参与生物被膜形成的具体生物学功能仍有待阐明。本研究基于成熟生物被膜的研究基础，进一步探究了rnc的作用。实验采用扫描电子显微镜（scanning electron microscopy）与结晶紫染色实验（crystal violet assay）检测菌株的生物被膜形成能力；通过胞外多糖染色分析生物被膜内胞外多糖（exopolysaccharides）的产生与分布情况；利用凝胶渗透色谱（gel permeation chromatography）开展分子量评估；通过原子力显微镜（atomic force microscopy）测定其黏附力；采用定量聚合酶链式反应（qPCR）在mRNA水平分析生物被膜形成相关基因的表达情况。本研究发现，在变形链球菌中，rnc可通过组装结构完整、被胞外多糖包裹的稳定生物被膜，参与生物被膜的形成并发挥功能。rnc缺陷菌株的生物被膜形成能力减弱，这与其胞外多糖产生减少及细菌黏附力降低相关。综合来看，本研究揭示了一个有趣的现象：此前未被充分认知的、与毒力相关的调控基因rnc，极有可能已被招募为变形链球菌（S. mutans）生物被膜形成的潜在调控因子。对rnc的进一步表征，有望为龋齿开发出靶向致病性生物被膜的特异性治疗手段。