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.