Analysis of Small RNAs in Streptococcus mutans under Acid Stress—A New Insight for Caries Research

Abstract

Streptococcus mutans (S. mutans) is the major clinical pathogen responsible for dental caries. Its acid tolerance has been identified as a significant virulence factor for its survival and cariogenicity in acidic conditions. Small RNAs (sRNAs) are recognized as key regulators of virulence and stress adaptation. Here, we constructed three libraries of sRNAs with small size exposed to acidic conditions for the first time, followed by verification using qRT-PCR. The levels of two sRNAs and target genes predicted to be bioinformatically related to acid tolerance were further evaluated under different acid stress conditions (pH 7.5, 6.5, 5.5, and 4.5) at three time points (0.5, 1, and 2 h). Meanwhile, bacterial growth characteristics and vitality were assessed. We obtained 1879 sRNAs with read counts of at least 100. One hundred and ten sRNAs were perfectly mapped to reported msRNAs in S. mutans. Ten out of 18 sRNAs were validated by qRT-PCR. The survival of bacteria declined as the acid was increased from pH 7.5 to 4.5 at each time point. The bacteria can proliferate under each pH except pH 4.5 with time. The levels of sRNAs gradually decreased from pH 7.5 to 5.5, and slightly increased in pH 4.5; however, the expression levels of target mRNAs were up-regulated in acidic conditions than in pH 7.5. These results indicate that some sRNAs are specially induced at acid stress conditions, involving acid adaptation, and provide a new insight into exploring the complex acid tolerance for S. mutans.

High-throughput sequencing identification and characterization of potentially adhesion-related small RNAs in Streptococcus mutans

“…[ 14 ]. Additionally, specific sRNAs of a small size (18–50 nucleotides) are specifically induced under acid stress conditions, as revealed in our previous studies [ 15 ]. However, all previous studies were performed with planktonic cells, and very little is known regarding the sRNAs obtained from attached cells, which exhibit different physiological properties from their planktonic counterparts [ 16, 17 ].…”

Phenotypes of Streptococcus mutans isolates in sound and cavitated tooth surfaces and its relationship to early childhood caries in China

“…Importantly, sRNAs coordinate adaptation processes in response to environmental changes in bacteria by regulating target mRNA expression [37][38][39][40][41]. Specifically, srn225147 and srn821798 were sequenced in our previous studies [22,23]. Virulence-related genes selected in our present study were predicted targets for these two sRNAs according to IntaRNA and RNAPredator.…”

“…The PCR reaction conditions for srn821798 were as follows: denaturation at 95°C for 10 s; 40 cycles consisting of 5 s of denaturation at 95°C; and 20 s of annealing at 50°C. The primers for 16S rRNA, gtfB, gtfC, spaP, srtA, ffh, and relA were synthesizedaccording to previous studies[8,[22][23][24].…”

Characterization of acid-tolerance-associated small RNAs in clinical isolates of Streptococcus mutans: Potential biomarkers for caries prevention

“…Our previous study (25) revealed that other sRNAs are differentially expressed within acidic environments. Future investigations aim to determine the sRNAs involved in the acid response exhibited by S. mutans, and to understand the molecular mechanisms and regulation of these molecules.…”

“…In our previous study, three libraries of sRNAs were constructed and exposed to acidic conditions (25). A total of 91 sRNAs with ≥1,000 mean reads were identified and nine candidates were randomly selected (Table I).…”