Exopolysaccharides metabolism and cariogenesis of Streptococcus mutans biofilm regulated by antisense vicK RNA

Abstract

Background: Streptococcus mutans (S. mutans) is a pivotal cariogenic pathogen contributing to its multiple virulence factors, one of which is synthesizing exopolysaccharides (EPS). VicK, a sensor histidine kinase, plays a major role in regulating genes associated with EPS synthesis and adhesion. Here we first identified an antisense vicK RNA (ASvicK) bound with vicK into double-stranded RNA (dsRNA).

Objective: This study aims to investigate the effect and mechanism of ASvicK in the EPS metabolism and cariogenesis of S. mutans.

Methods: The phenotypes of biofilm were detected by scanning electron microscopy (SEM), gas chromatography-mass spectrometery (GC-MS) , gel permeation chromatography (GPC) , transcriptome analysis and Western blot. Co-immunoprecipitation (Co-ip) assay and enzyme activity experiment were adopted to investigate the mechanism of ASvicK regulation. Caries animal models were developed to study the relationship between ASvicK and cariogenicity of S. mutans.

Results: Overexpression of ASvicK can inhibit the growth of biofilm, reduce the production of EPS and alter genes and protein related to EPS metabolism. ASvicK can adsorb RNase III to regulate vicK and affect the cariogenicity of S. mutans.

Conclusions: ASvicK regulates vicK at the transcriptional and post-transcriptional levels, effectively inhibits EPS synthesis and biofilm formation and reduces its cariogenicity in vivo.

Keywords: Antisense RNA; Streptococcus mutans; bacterial genetics; biofilm growth; dental caries; exopolysaccharides; vicK gene.

Figure 1.

ASvicK altered morphology and growth of bacteria and biofilm. (a) Morphology structure of UA159, ASvicK and SmuvicK+. Significant differences were apparent in ASvicK strain, which has a longer chain and diverse size and shapes; (b) SEM of UA159, ASvicK, and SmuvicK+ cultured in BHI supplemented with 1% sucrose. The ASvicK strain appeared longer and diverse cell size and shape; (c) Growth curve of UA159 and the vicK mutant strain. The bacteria strains were grown in BHI at 37°C anaerobically and were monitored every hour; (d) CFU curve of UA159 and the vicK mutant strain. The bacteria strains were grown in BHIS at 37°C anaerobically and were monitored every 4 hours (h); (e) the biofilm growth of S. mutans obtained from crystal violet assay experiments revealed the ASvicK strain caused a reduction in biofilm formation. The results were averaged from 8 independent cultures of different strains (UA159, ASvicK, and SmuvicK+), and experiments were performed in triplicate (n = 3; *: p < 0.05); (f) the capacity of lactic acid production of biofilm were determined. The ASvicK strain had the lowest capacity of lactic production. The results were averaged from 8 independent cultures of different strains (UA159, ASvicK, and SmuvicK+), and experiments were performed in triplicate (n = 3; *: p < 0.05); (g) LDH activity were determined which revealed that the ASvicK strain had the lowest activity (n = 3; *: p < 0.05); (h) SEM of biofilms of UA159, ASvicK, and SmuvicK+ cultured in BHI supplemented with 1% sucrose. The ASvicK strain lower amounts of bacteria and EPS and looser biofilm structure. The yellow arrows represent bacteria while the red arrows represent EPS; (i) LDH activity/CFU were determined which revealed that the ASvicK strain had the lowest activity (n = 3; *: p < 0.05); (j) Lactic acid production/CFU of biofilm were determined. The ASvicK strain had the lowest capacity of lactic production (n = 3; *: p < 0.05).

Figure 2.

ASvicK inhibits EPS production in vitro. (a) CLSM of biofilms of UA159, ASvicK, and SmuvicK+ cultured in BHI supplemented with 1% sucrose. The ASvicK strain lower amounts of bacteria and EPS and looser biofilm structure and experiments were performed in triplicate; (b) Production of WIGs and WSGs of strains were measured by anthrone–sulfuric acid colorime tric assay. The results were averaged from 8 independent cultures of different strains (UA159, ASvicK, and SmuvicK+), and experiments were performed in triplicate (n = 3; *: p < 0.05); (c) a monosaccharide composition analysis of the polysaccharide was carried out. The results indicated the polysaccharide comprised Glc, Gal and Man with various molar ratios; (d) the molecular weight distribution of the polysaccharides of samples from different strains was estimated using GPC.

Figure 3.

ASvicK alters genes and enzymatic activity related to exopolysaccharide metabolism. (a) the classification and percentage of the UA159 and the ASvicK strain DEGs were analyzed according to their functional annotations; (b) Gene ontology enrichment analysis of the DEGs using the DAVID tool. Upregulated genes are shown in red, and downregulated genes are shown in green; (c) RT-qPCR analysis showed the gene related to EPS metabolism transcripts in the UA159, ASvicK, and SmuvicK+ strains. S. mutans gene expression was relatively quantified by RT-qPCR using gyrA as an internal control (n = 3; *: p < 0.05); (d) RT-qPCR analysis showed the gene related to lactose and galactose metabolism transcripts in the UA159, ASvicK, and SmuvicK+ strains. S. mutans gene expression was relatively quantified by RT-qPCR using gyrA as an internal control (n = 3; *: p < 0.05); (e) the effect of ASvicK on GtfB/C/D enzymatic activity was verified by zymogram analysis.

Figure 4.

ASvicK regulates the expression of vicK on post-transcriptional level. (a) the production of VicK and Rnc was quantified by Western blotting in the cells grown; (b) Production of recombinant Rnc and RNase III activity assays. Equal amounts of RNA (2 µg) were incubated with 4 nM recombinant Rnc in 20 µL interaction buffer (10 mM Tris-HCl, pH 8.0) for 30 min at 37°C. For controls, equal amounts of RNA (2 µg) were incubated in 20 µL interaction buffer (10 mM Tris-HCl, pH 8.0) for 30 min at 37°C. M: Marker; Lane 1: UA159 total RNA + reaction mixture; lane 2: ASvicK strain total RNA + reaction mixture; lane 3: SmuvicK+ strain total RNA + reaction mixture; lane 4: UA159 total RNA + recombinant Rnc; lane 5: ASvicK strain total RNA+ recombinant Rnc; lane 6: SmuvicK+ strain total RNA+ recombinant Rnc; lane 7: UA159 total RNA; lane 8: ASvicK strain total RNA; lane 9: SmuvicK+ total RNA; (c) the post-transcriptional regulation mechanism of ASvicK was detected by co-ip. The expression of msRNA1657 was detected by RT-qPCR; (d) the expression of RNase III was detected by co-ip. ASvicK can enrich RNase III and form RNA-protein complex; (e) Working model of regulation by ASvicK.

Figure 5.

ASvicK suppresses cariogenic pathogenicity in vivo. (a) RT-qPCR analysis of ASvicK RNAs of clinical strains dental plaque from SECC children and CF children. S. mutans gene expression was relatively quantified by RT-qPCR and calculated based on UA159 expression set as 1.0 with gyrA as an internal control. Experiments were performed in triplicate and presented as the mean ± standard deviation; Shapiro–Wilk tests and Bartlett tests showed that the data were non-parametric. Significant differences were determined using the Kruskal–Wallis test and least significant difference (LSD) multiple comparisons method (n = 8; *: p < 0.05); (b) Keyes scores were used to calculate the pit and fissure caries, the smooth surface caries and total caries respectively in experimental rat. Shapiro-Wilk tests and Bartlett tests showed that the data were non-parametric. Significant differences were determined using the Kruskal–Wallis test and LSD multiple comparisons method (n = 8; *: p < 0.05); (c) the severity of pit fissure caries lesions of rat molars was observed under stereomicroscope; (d) SEM of biofilms on rat molars. Biofilm in the ASvicK group has significantly reduced EPS matrix similar to the blank control group, while the UA159 group and the SmuvicK+ group were covered with abundant EPS matrix; (e) Double labeling of biofilms on the tooth surface of rat molars. Green, total bacteria (SYTO 9); red, EPS (Alexa Fluor 647); scale bars, 100 µm. The ASvicK group has less bacteria and EPS matrix similar to the blank control group than the UA159 and SmuvicK+ group.