Carbohydrate Metabolism Regulated by Antisense <i>vicR</i> RNA in Cariogenicity

Abstract

<i>Streptococcus mutans</i> is a major cariogenic pathogen that resides in multispecies oral microbial biofilms. The VicRK 2-component system is crucial for bacterial adaptation, virulence, and biofilm organization and contains a global and vital response regulator, VicR. Notably, we identified an antisense <i>vicR</i> RNA (AS<i>vicR</i>) associated with an adjacent RNase III-encoding (<i>rnc</i>) gene that was relevant to microRNA-size small RNAs (msRNAs). Here, we report that ASvicR overexpression significantly impeded bacterial growth, biofilm exopolysaccharide synthesis, and cariogenicity in vivo. Transcriptome analysis revealed that the AS<i>vicR</i> RNA mainly regulated carbohydrate metabolism. In particular, overproducing AS<i>vicR</i> demonstrated a reduction in galactose and glucose metabolism by monosaccharide composition analysis. The results of high-performance gel permeation chromatography revealed that the water-insoluble glucans isolated from AS<i>vicR</i> presented much lower molecular weights. Furthermore, direct evidence showed that total RNAs were disrupted by <i>rnc</i>-encoded RNase III. With the coexpression of T4 RNA ligase, putative msRNA1657, which is an <i>rnc</i>-related messenger RNA, was verified to bind to the 5'-UTR regions of the <i>vicR</i> gene. Furthermore, AS<i>vicR</i> regulation revealed a sponge regulatory-mediated network for msRNA associated with adjacent RNase III-encoding genes. There was an increase in AS<i>vicR</i> transcript levels in clinical <i>S. mutans</i> strains from caries-free children, while the expression of AS<i>vicR</i> was decreased in early childhood caries patients; this outcome may be explored as a potential strategy contributing to the management of dental caries. Taken together, our findings suggest an important role of AS<i>vicR</i>-mediated sponge regulation in <i>S. mutans</i>, indicating the characterization of lactose metabolism by a vital response regulator in cariogenicity. These findings have a number of implications and have reshaped our understanding of bacterial gene regulation from its transcriptional conception to the key roles of regulatory RNAs.