Small noncoding RNA sRNA0426 is involved in regulating biofilm formation in Streptococcus mutans

Abstract Evidence suggests that small noncoding RNAs (sRNAs) are involved in the complex regulatory networks governing biofilm formation. Few studies have investigated the role of sRNAs in Streptococcus mutans (S. mutans). In the present study, the association between sRNA and biofilm formation in S. mutans was explored. sRNAs that are differentially expressed in the biofilm and planktonic states of this bacterium were identified by quantitative real‐time PCR (qRT‐PCR). Confocal laser scanning microscopy was used to investigate the characteristics of biofilm formation in a standard strain of S. mutans (UA159, ATCC 700610) and ten clinical strains. Bioinformatics analyses were employed to predict and examine potential sRNA regulatory pathways. The results showed that sRNA0426 has a strong positive relationship with dynamic biofilm formation. Moreover, sRNA0426 expression was positively correlated with exopolysaccharide (EPS) production. Bioinformatics analyses showed that sRNA0426 is involved in biofilm formation such as metabolic pathways, especially carbon metabolism. Five target mRNAs (GtfB, GtfC, GtfD, ComE, and CcpA) involved in the synthesis of EPS were selected for further evaluation; the expression levels of three of these mRNAs (GtfB, GtfC, and CcpA) were positively correlated with sRNA0426 expression levels, and the expression level of one (ComE) was negatively correlated. In conclusion, the results suggested that sRNA0426 may play an important and positive role in the biofilm formation of S. mutans and provide novel insight into the S. mutans biofilm regulatory network.

For example, Zhao, Koestler, Waters, and Hammer (2013) found that Qrr sRNAs simultaneously negatively and positively regulate expression of the hapR gene and the vca0939 gene, respectively, to promote biofilm formation in Vibrio cholera. In Streptococcus sanguinis, two sRNAs that negatively regulate biofilm formation by inhibiting the expression of the target pilT gene were recently identified (Ota et al., 2018).
Additionally, Lee and Hong (2012) revealed more than 900 sRNAs and highlighted the importance of sRNAs in S. mutans. In a previous study, we established a library of 736 differentially expressed candidate sRNAs associated with initial adhesion in S. mutans UA159 by RNA deep sequencing (Zhu, Liu, Liu, Zhou, & Lin, 2018). Moreover, we observed a consistent correlation between the expression of sRNAs and initial adhesion ability in 100 clinical strains of S. mutans (Zhu et al., 2018). Initial adhesion is the first step in biofilm formation, the processes of which include reversible attachment, irreversible attachment, maturation, and dispersion (Hinsa, Espinosa-Urgel, Ramos, & O'Toole, 2003). The microbial composition and structure change dynamically during biofilm formation.
Although sRNAs are widely considered to act as key regulators in biofilm formation (Svenningsen, 2018), there has been limited investigation of the role of these molecules in the dynamic process of biofilm formation (Kreth, Liu, Chen, & Merritt, 2015), and it remains unknown whether sRNA exerts an important role during the process of biofilm formation in S. mutans.
In this study, we first screened sRNAs associated with biofilm formation in the standard strain of S. mutans UA159 and then investigated the potential association between sRNAs and biofilm formation and the production of exopolysaccharide (EPS) in clinical strains of S. mutans. Bioinformatics analysis was used to predict and verify the potential regulatory mechanisms employed by candidate sRNAs. The results highlight the function of sRNAs in the dynamic regulation of biofilm formation and provide a promising avenue for developing novel methods of caries prevention by targeting S. mutans.

| Bacterial strain and culture conditions
The strains used in the present study included the standard strain of S. mutans (UA159ATCC 700610) and clinical isolates. Clinical isolates were obtained from an epidemiological survey conducted in Guangdong Province, People's Republic of China, in 2015 (Yu et al., 2015). The survey was conducted among 5-year-old children. A total of 215 clinical strains were isolated from 215 children with different caries status (Zhu et al., 2018). From these isolates, 10 clinical strains were randomly selected. The S. mutans strains were grown in brain heart infusion (BHI) broth (Oxoid) overnight under anaerobic conditions (80% N 2 , 20% CO 2 ) at 37°C. The optical density at 600 nm (OD 600 ) of overnight-cultured strains was measured using a microplate reader (Bio-tek, Epoch 2, America). UA159 suspensions (OD 600 = 0.7) were inoculated at 1:20 into fresh BHI in round-bottom 6-well plates to obtain planktonic cells; the same suspensions (OD 600 = 0.7) were inoculated at 1:20 into fresh BHI in flat-bottom 6-well plates and incubated for 4 h, 6 h, 12 h, and 24 h to monitor the dynamic biofilm formation process of S. mutans.

| RNA extraction
Planktonic bacteria were collected by centrifugation at (13201 g) for 5 min. Biofilm bacteria were scraped from plates and centrifuged at (13201 g) for 5 min. Total RNA extraction was performed according to the method described by Zhu et al. (2017). Briefly, total RNA was stabilized with RNAprotect Bacteria Reagent (Qiagen) before extraction. Biofilms were harvested and washed three times with phosphate-buffered saline (PBS). The total RNA of biofilm cells was purified using a miRNeasy Mini Kit (Qiagen). A Thermo Scientific NanoDrop 2000 instrument (NanoDrop Technologies) and an Agilent 2100 system (Agilent Technologies) were used to assess RNA quality and quantity.

| Quantitative real-time PCR (qRT-PCR)
The top twenty significantly differentially expressed sRNAs were selected as candidates from our sRNA library established in a previous study (Zhu et al., 2018). These candidate sRNAs were further analyzed between planktonic and biofilm conditions of S. mutans at 24 h by qRT-PCR. cDNA was synthesized using a Mir-X miRNA First- The most highly expressed sRNA associated with biofilm formation from the analyzed 20 sRNAs was selected for further analysis. Five target mRNAs of the candidate sRNA were selected, and their possible functional roles were preliminarily explored. The primers used in the qRT-PCR analysis of the mRNAs are listed in Table 2. Synthesis of cDNA and qRT-PCR were performed as described above. The expression level of each gene was determined in triplicate. Expression levels were calculated using the 2−ΔΔ Ct method (Livak & Schmittgen, 2001).

| Crystal violet (CV) staining assay
The CV staining assay was used to evaluate the biofilm biomass of S. mutans (Weerasekera et al., 2016).

| Confocal laser scanning microscopy (CLSM)
For analysis of EPS production, 1 μM Alexa Fluor 647 (Invitrogen) and 2.5 μM SYTO 9 (Invitrogen) were used to label dextran and bacterial cells, respectively (Huang et al., 2017). COMSTAT was used to analyze the biomass of EPS (μm 3 /μm 2 ). The three-dimensional architecture of the biofilms was reconstructed using Imaris 8.0.2 (Bitplane). Three independent experiments were performed for each condition, and images of five random fields were collected for each sample.

| Bioinformatics analysis of candidate sRNAs
We predicted the structures of candidate sRNA using RNAfold

| Statistical analyses
Each experiment was independently repeated three times. GraphPad

| Screening for the most highly differentially expressed sRNAs associated with biofilm formation
To obtain the most relevant sRNAs associated with biofilm formation, we screened the top 20 differentially expressed sRNAs from our previous study in the standard strain of S. mutans (UA159, ATCC 700610). Among the 20 sRNAs, 18 were successfully detected with 14 were upregulated in cultures with biofilm status relative to those with planktonic status, and 4 were downregulated. Two sRNAs (sRNA0250 and sRNA0656) expressed so unstably after multiple repeated studies that the detection of these two sRNAs was not shown in Table 3. sRNA0426 was the most highly differentially expressed sRNA. Its expression was 5.87 times higher in the biofilm state than in the planktonic state (p < 0.001, Table 3).

| Expression of sRNA0426 during biofilm formation in standard and clinical strains of S. mutans
To further verify the relationship between sRNA0426 and biofilm formation, we first evaluated the biofilm biomass by CV assays and

| Relationship between expression of sRNA0426 and EPS
EPS forms the core of the matrix scaffold and provides binding sites that promote the accumulation of microorganisms on the tooth surface and the establishment of pathogenic biofilms (Bowen, Burne, Wu, & Koo, 2018). Thus, to further explore the association between sRNA0426 and EPS, we specifically analyzed EPS by CLSM.
According to the confocal micrographs of EPS, the bacteria became These results indicate that sRNA0426 may play a positive role in the production of EPS in S. mutans biofilms.

| Functional annotation of sRNA0426 using bioinformatics analyses
Considering the importance of secondary structures in stabilizing sRNAs, the secondary structure of sRNA0426 was predicted using RNAfold. It is reported that sRNA0426 possesses a stem-loop structure with a ΔG value of −18.7 kcal/mol (Figure 5a). To the best of our knowledge, sRNA0426 is located on the antisense mRNA strand between SMU_1238c and SMU_1239 (Table A1). To explore the potential mechanism by which sRNA0426 regulates S. mutans biofilm formation, KEGG pathway annotation was used to investigate the sRNA0426 regulatory pathway, revealing eight pathways that are significantly regulated by sRNA0426 (p < 0.05) (Figure 5b) Note: Expression of the 20 selected sRNAs under standard Streptococcus mutans biofilm conditions compared with planktonic conditions at 24 h. 18 sRNAs were differentially expressed between the two conditions; of these, sRNA0426 was the most significantly upregulated sRNA. The expression of sRNA0250 and sRNA0656 under these two conditions was not measured stably. The sequencing data for these sRNAs were obtained from (Zhu et al., 2018). To determine whether similar putative sRNAs are present in other bacteria, we searched for sequences homologous of sRNA0426 using BLASTN. The results are shown in Figure 6. A sequence was only considered to be conserved when the coverage between the query and subject sequences was higher than 75% and the nucleotide identity was higher than 65% (E-value = 10 −5 , word = 11). The

| Relationship of sRNA0426 and potential target mRNAs
To further explore the function of sRNA0426, we examined the association between sRNA0426 and five potential target mRNAs predicted by bioinformatics (GtfB, GtfC, GtfD, ComE, and CcpA) at 12 h, when the strongest correlation was observed between sRNA0426 and biofilm biomass together with EPS. According to the results, sRNA0426 expression showed a significantly positive relationship with GtfB, GtfC, ComE, and CcpA expression (p < 0.05) but no significant relationship with GtfD expression (Figure 7). Potential binding sites were also predicted by intaRNA ( Figure A1). The presence of binding sites between potential target mRNAs and sRNA0426 provides evidence for a regulatory role of sRNA0426.

| D ISCUSS I ON
Biofilm formation of S. mutans is a dynamic process that involves biofilm-specific genetic mechanisms and regulatory networks that allow the bacterium to adapt to a changing microenvironment  (Shemesh, Tam, & Steinberg, 2007). In our study, the expression of sRNA0426 was significantly higher in biofilms than in the planktonic state, and it changed dramatically during the biofilm formation process, showing a strong association with biofilm formation. Together with the correlation between sRNA0426 and biofilm biomass, the data suggest that sRNA0426 is associated with biofilm formation in S. mutans.
Biofilms are highly dynamic and structured communities of bacteria enmeshed in a self-produced matrix of extracellular polymeric substances (Flemming & Wingender, 2010;Flemming et al., 2016).
EPS forms the core of the matrix scaffold and provides a binding site for bacterial cells, mediating their adherence to form mature biofilms (Koo, Falsetta, & Klein, 2013). As attractive and effective regulators, cules to determine EPS production in Yersinia pestis (Liu et al., 2016).
Additionally, sRNAs cooperate with Hfq to regulate EPS production in Erwinia amylovora (Zeng, McNally, & Sundin, 2013). In the present study of S. mutans, sRNA0426 displayed a positive correlation with EPS. The results suggest that sRNA0426 plays an important role in S. mutans biofilm formation through the production of EPS.

Synthesis of EPS is determined by carbon metabolism, which in
S. mutans is mainly controlled by glucosyltransferases (gtfs). GtfBC metabolizes sucrose to produce water-insoluble glucans, and gtfD synthesizes predominantly soluble glucans to establish the EPS matrix (Li & Burne, 2001). The activities of gtfs are controlled by regulators. For example, comE is part of two-component signal transduction systems and it is an occluded RNA polymerase that binds to the coding region of gtfC to abort its expression, thereby interfering with carbon metabolism and biofilm formation (Hung et al., 2011). Furthermore, ccpA plays a critical role in the response to carbon source availability by affecting the stability of biofilms in S. mutans, and the gtfBC genes require ccpA for optimal expression (Wen & Burne, 2002).
In general, sRNAs regulate gene expression by base-pairing with target mRNAs or by binding proteins directly (Chambers & Sauer, 2013) Associations between sRNA0426 and target mRNAs, including GtfB, GtfC, ComE, and CcpA, were examined, and the results further supported the role of sRNA0426 in the production of EPS biomass. The positive correlation between GtfB, GtfC, CcpA, and sRNA0426 expression, together with the negative correlation between ComE and sRNA0426, suggest that sRNA0426 might be positively associated with biofilm formation in the regulation of EPS.
KEGG analysis of the predicted target genes of sRNA0426 suggests that sRNA0426 is involved in diverse physiological activities through 8 pathways (p < 0.05), such as metabolic pathways including carbon metabolism and microbial metabolism in diverse environments, that are associated with biofilm formation. What's more, the seed sequence is necessary but insufficient (Didiano & Hobert, 2006;Lee et al., 2016). And the most stable predicted binding sites between the biofilm-associated mRNAs and sRNA0426 are not limited in the seed sequence. Therefore, the seed sequence of sRNA0426 might serve an important role for sRNA0426, but the association between it and the function of sRNA0426 in S. mutants on biofilm formation is needed to be further verified (Fritsch, Siqueira, & Schrank, 2018).
Overall, the functions of sRNAs may be more complex than once considered. The present study is a primary exploration of biofilm-associated sRNAs in S. mutans. The identification of more potential sRNAs and function analysis of additional sRNAs are required, and especially creating mutans to further analyze the role of sRNAs in S. mutans is needed. We have tried and failed to create mutant strains. The details are described in the Appendix A and shown in Figure A2. This could point to an essential function of this sRNA or more attempts to try.
In conclusion, we first explored the expression characteristics and potential functions of sRNAs in the biofilm formation process of standard S. mutans and clinical strains. We found that sRNA0426 and its target mRNAs are dynamically involved in the synthesis of EPS and biofilm-associated pathways. The results presented herein

F I G U R E 7
The potential role of sRNA0426 in biofilm formation. (a-e) The level of expression of sRNA0426 and potential target mRNAs in strain 5521 was defined as 1.0. Spearman correlation analysis of sRNA0426 expression with ComE, GtfBCD, and CcpA is shown in the figure for the 10 clinical isolates at 12 h suggest the presence of a novel regulator in S. mutans under biofilm conditions, providing a better understanding of the mechanism of biofilm formation.

This work was supported by grants from the National Natural
Science Foundation of China (Grant No. 81570967).

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
The study protocol was approved by the Ethics Committee of the Guanghua School of Stomatology, Sun Yat-sen University (ERC-

CO N S TRU C TI O N O F TH E S R N A0 426 D E LE TI O N
Considering the length of sRNA0426 and the situation that there was no referable study of sRNAs deletion in Streptococcus mutans, we decided to attempt to construct the sRNA0426 deletion by markerless mutagenesis (Geng et al., 2016;McDaniel, Mackay, Quiroz, & Chilkoti, 2010). The knockout cassette for double-crossover homologous recombination was amplified from p15A (Selzer, Som, Itoh, & Tomizawa, 1983), named pTG17301. pTG17301 contains a counter selectable marker (phes) (Liu et al., 2020) and antibiotics marker that works in both Gram-negative and Gram-positive bacteria and will not lead to polar effects. Then, primers sRNA0426 LF and sRNA0426 RF were used to amplify the left and right flanks of sRNA0426 from the regions adjacent to its coding region of S. mutans UA159, generating 971-bp and 992-bp amplicons. The left flank region and the right flank region were ligated to pTG17301 by seamless ligation (Tolobio Ezmax one-step cloning Kit), creating plasmid pTC17316. And the left flank region and the right flank region were also ligated together by seamless ligation to replace the knockout cassette. The gene replacement vector was transformed into S. mutans UA159 in the presence of 1 μg/μl competence-stimulating peptide. Following the allelic exchange, S. mutans UA159 with sRNA0426 deficiency was isolated on BHI and chemically defined medium (CDM) (van de Rijn & Kessler, 1980) agar plates, with or without exogenous d-Glu (30 mM), supplemented with 100 μg/ml antibiotics and further confirmed using colony Polymerase chain reaction (PCR) and agarose gel electrophoresis (AGE).

CO N S TRU C TI O N FA I LU R E O F TH E S R N A0 426 D E LE TI O N
We carried many attempts to construct mutant strains, such as circular plasmid and linear plasmid transformation by chemical transformation and electrotransformation under different conditions. All the results were negative. In the control plates of S. mutans UA159, no strain was grown ( Figure A2a). In the vast majority transformation plates, almost no possible deletion strain was grown ( Figure A2b). And in the very few transformation plates strain, a small number of possible deletion strains were grown ( Figure A2c). In confirmation of AGE, the lane of the wild type was located at 2000 bp, while the positive colony should be located at 4000 bp. Although there are some potential positive colonies in the transformation plates, the results suggested that only the bands of 2000 bp were detected, which meant the verified strains were all false positive ( Figure A2d).

D I SCUSS I O N
We have tried and failed to construct a sRNA0426 mutant strain for S. mutans. This could point to an essential function of sRNA0426 or more attempts to improve the experimental methods and more trials. The present results might suggest a possibility of an important role of sRNA0426, and another situation is that homologous recombination is a probabilistic event where the times of repetitions were not enough under the tough condition of the length of sRNA0426 together with its stability. We might make a further try and confirmation of the role of sRNA0426 in the future. Note: The data are extracted from Zhu et al. (2018). AM indicates that the sRNA is located on the antisense strand to mRNA; IGR indicates that the sRNA is in the intergenic region.