Dissecting the essential role of luxS-relevant small RNAs in biofilm formation of Streptococcus mutans

Streptococcus mutans is considered the key contributor to human dental caries. The LuxS/AI-2 quorum sensing (QS) system in S. mutans plays a crucial role in the development of cariogenic oral biofilms. Many QS systems rely on regulation carried out by small RNAs (sRNAs) to achieve optimal performance. However, the identities and functions of sRNAs in S. mutans QS system are not well elucidated. Here, we identify two novel sRNAs named SmqsR2 and SmqsR4 (Streptococcus mutans quorum-sensing related small RNA 2 and 4), which respond to luxS expression and support biofilm formation by mediating exopolysaccharides (EPS) synthesis. We showed that overexpression of SmqsR2 and SmqsR4 in S. mutans, respectively, resulted in declined growth rates, altered biofilm architecture, increased EPS production, and upregulated expression of gtfB/C/D genes. Transcriptome analysis revealed that several genes related to carbohydrate utilization were differentially expressed, with ccpA being markedly upregulated in both SmqsR2 and SmqsR4 overexpression strains. Specifically, SmqsRs promoted ccpA transcript, and CcpA triggered transcription of SmqsR2 and SmqsR4 via direct binding, thus forming a positive feedback loop. We speculate that SmqsRs augment LuxS-mediated biofilm matrix production, most likely through the activation of gtfB/C/D expression and the reprogramming of S. mutans central carbon metabolism by ccpA. In summary, we have confirmed that SmqsR2 and SmqsR4 function as supporting factors that maintain the optimal status of the LuxS/AI-2 system in S. mutans, which is important for cell growth, EPS synthesis, and biofilm formation. Hence, sRNA activity may represent a promising target to modulate S. mutans cariogenicity.

变形链球菌（Streptococcus mutans）被认为是人类龋齿的关键致病因素。其体内的LuxS/AI-2群体感应（Quorum Sensing，QS）系统在致龋口腔生物被膜的形成过程中发挥关键作用。诸多群体感应系统依赖小RNA（sRNAs）介导的调控以实现最佳功能，但目前学界对变形链球菌群体感应系统中小RNA的具体身份与功能尚未充分阐明。本研究鉴定出两种新型小RNA，命名为SmqsR2与SmqsR4（即变形链球菌群体感应相关小RNA2和4），它们可响应luxS基因的表达，并通过介导胞外多糖（EPS）合成促进生物被膜形成。实验结果显示，分别在变形链球菌中过表达SmqsR2与SmqsR4，会导致菌株生长速率下降、生物被膜结构改变、胞外多糖产量提升，以及葡萄糖基转移酶B/C/D（gtfB/C/D）基因的表达上调。转录组分析表明，多个与碳水化合物利用相关的基因存在差异表达，且在SmqsR2与SmqsR4过表达菌株中，分解代谢物控制蛋白A（ccpA）基因均显著上调。具体而言，SmqsRs可促进ccpA的转录本积累，而CcpA则通过直接结合触发SmqsR2与SmqsR4的转录，由此形成正反馈环路。本研究推测，SmqsRs可增强LuxS介导的生物被膜基质合成，这一过程极有可能通过激活gtfB/C/D的表达，以及通过ccpA重编程变形链球菌的中央碳代谢途径实现。综上，本研究证实SmqsR2与SmqsR4作为辅助因子，可维持变形链球菌中LuxS/AI-2群体感应系统的最佳状态，这对于菌株生长、胞外多糖合成以及生物被膜形成均具有重要意义。因此，小RNA的调控活性有望成为干预变形链球菌致龋性的潜在靶点。