3, 5-Di-tert-butylphenol Combat Against Streptococcus Mutans Via Impedes Acidogenicity, Acidurance and Biolm Formation

Streptococcus mutans is a common pathogen present in oral cavity and it cause dental caries for all aged group of people, in particular, children’s. S. mutans have several virulence factors such as acidogenecity, aciduricity, adhesion and biolm formation. These virulence factors are working together and lead to initiate development of caries in tooth surface. The present study aimed to investigate the anticariogenic potential of 3, 5-Di-tert-butylphenol (3, 5-DTBP) against S. mutans. 3, 5-DTBP biolm inhibitory concentration (BIC) was found at 100 µg/ml concentration without any lethal effect on the growth. Moreover, 3, 5- DTBP signicantly reduced water soluble and water insoluble glucans production, in concurrence with downregulation of gtfBC genes. Moreover, acidogenicity associated virulence factors such as lactate dehydrogenase and enolase enzymatic production was arrested upon 3, 5-DTBP treatment. In addition, 3, 5-DTBP greatly reduced acidtolerance ability through impedes of F 1 F 0 -ATPase. Gene expression analysis unveiled the downregulation of gtfB, gtfC, gtfD, vicRK, comDE, gbpB, smu0630 and relA upon 3, 5-DTBP treatment. The present study paves the way for exhibiting 3, 5-DTBP as a promising therapeutic agent to control S. mutans infections.


Introduction
Dental caries is one of the leading oral infectious diseases caused by Streptococcus mutans (Marsh 2004). Streptococcus mutans is a primary causative agent since its adeptness to extreme environmental stress conditions while bio lm formation and host interactions (Smith and Spatafora 2012). S. mutans have equipped multiple factors which assist them to adhere, interact with nearby microbiota which leads to initiate the progression of infection ). S. mutans cariogenic virulence and survival ability were controlled by two component signal transduction systems (TCSTS). VicRK is well de ned TCSTS, controlled various virulence factors and physiological progress of S. mutans such as bio lm formation, regulate gtfBCD expression, acid tolerance, oxidative stress tolerance, genetic competence and cell death (Senadheera et al. 2005;Deng et al. 2007; Mattos-Graner and Duncan 2017). ComDE is another virulence gene, regulates gene competence and bio lm formation (Li et al. 2002). In the cooperation of these genes function, assist S. mutans to adhere/colonization on the tooth surface which initiate caries development progress (Martins et al. 2018). S. mutans synthesis various organic acids (pH range between 3-5 pH) done by the carbohydrate fermentation process (Matsui and Cvitkovitch 2010). S. mutans have survived on a low pH environments; F 1 F 0 -ATPase is involved to pump the protons from the cell and maintains the inner cell pH level (Lemos and Bume 2008). In addition, bio lm formation keeps control of the antimicrobials penetration, it acts as a primary barrier to protect the bacterial cells during a low pH environment. In particularly mature bio lm cells are high acid tolerance compared to planktonic cells (Welin-Neilands and Svensäter 2007). For their regular usage of antiseptic drugs for oral infections, it could cause severe to mild side effects such as teeth staining and hypersensitivity (Bagis et al. 2011).
Hence, the new antimicrobial agents/alternative therapeutic action is needed to control oral infections in clinical settings.
Plants are a promising source for identifying antimicrobial agents with minimal side effects, compared to commercial antibiotics and other sources. This property added additional value for plants sources to produce appropriate antimicrobial agents for clinical applications against infectious diseases (Gyawali and Ibrahim 2014). Several antimicrobial compounds have been discovered from plant sources and many of them are commercially available, with some compounds already have been used for caries prevention (Cardoso et al. 2016; Vijayaumar and Ramanathan 2018; Zhang et al. 2020). The present study is aimed to unveiling the antibio lm and anticariogenic e cacy of organic compound 3, 5-di-tertbutylphenol (3, 5-DTBP) against a primary etiological pathogen Streptococcus mutans and to reveal the mechanism underlying its anticariogenic activity.

Materials And Methods
Bacterial strains and growth conditions Streptococcus mutans UA159 (reference strain) from American Type Culture Collection (ATCC 700610) was used in the study. It was maintained on tryptone soya agar (TSA) and cultured regularly in tryptone soya broth (TSB) at 37° C for 24 h. The standard cell suspension of S. mutans for all in vitro experiments was prepared by adjusting the optical density (OD) of overnight culture of 0.4 at 600 nm (1 x 10 8 CFU/ml) using TSB. Note: In all the assays performed in TSB supplemented with 1 % sucrose.
Screening of phytochemicals antibio lm e cacy against S. mutans Totally ve phytochemicals were screened for their antibio lm activity using 24 well sterile microtitre plates. Each well contains 1 ml of TSB (supplemented with 1 % sucrose) with 1 % of overnight culture bacterial culture (1 x 10 8 CFU/ml). For screening, 100 µg/ml of each compound was loaded and assay plate was kept at 37° C for 24 h. After incubation, OD at 600 nm was measured. Then planktonic cells were discarded and each well was washed gentle with sterile distilled water to eliminate loosely bound cells and then the plate was allow to air dried. For quanti cation, bio lm cells were stained with 0.4 % of crystal violet (CV) for 10 min and excess stain was removed by washing plates thrice with distilled water and air dried for 5 min. Bio lms cells were destained using 20 % glacial acetic acid and OD was read at 570 nm (Vijayakumar and Ramanathan 2020a).

Microscopic analysis of bio lm
In microscopic analysis, bacterial cells were allowed to form bio lm on glass slides (1 x 1 cm) in the presence and absence of 3, 5-DTBP for 24 h at 37° C. After incubation, the slides were washed with sterile distilled water and stained as needed. For light microscopic analysis, the washed slides were stained with 0.4 % CV and washed to remove excess stain and allow to air dried. Then, the glass slides were visualized at magni cation of 400X under light microscope. For confocal laser scanning microscope (CLSM) analysis, the washed slides were stained with 0.1 % of acridine orange for 10 min at dark followed by washing to remove the excess stain and drying. Finally, the glass slides were visualized under CLSM at magni cation of 200X (Vijayakumar et al. 2021).

Effect of 3, 5-DTBP on adherence to glass surface
The effect of 3, 5-DTBP on the sucrose-dependent and sucrose independent adherence of S. mutans over the smooth glass surface was performed as described earlier ). Brie y, S. mutans was grown anaerobically at 37° C at an angle of 30° in boiling test tubes containing 10 ml of TSB with or without 5 % sucrose in the presence and absence of 3, 5-DTBP and incubated for 24 h. After incubation, the planktonic cell suspension was removed and the adhering cells were washed by adding 0.5 M of sodium hydroxide followed by agitation. Then, the adhering cells were washed and suspended in PBS saline and the adherence was quanti ed using a spectrophotometer at OD 600 nm. The percentage of adherence was calculated according to the formula: Adherence (%) = (OD of adhered cells/OD of total cells) x 100 Effect of 3, 5-DTBP on cell surface hydrophobicity (CSH) of S. mutans To evaluate the effect of 3, 5-DTBP on the CSH of S. mutans was performed by microbial adhesion to hydrocarbon (MATH) assay as described earlier with slight modi cations (Khan et al. 2010). Brie y, overnight cultures grown in TSB in the presence and absence of 3, 5-DTBP were washed and suspended with sterile saline (0. 85 %) and their optical density (OD) as 0.3 at 600 nm. The cell suspension (3 ml) was taken to the test tubes containing 0.25 ml of toluene. Then, the tubes were agitated uniformly for 2 min and let to equilibrate for 10 min at room temperature. After separation of the toluene phase from the aqueous phase, the OD of the aqueous phase was measured using a spectrophotometer at 600 nm. The percentage of hydrophobicity was calculated according to the formula: % of hydrophobicity = [Initial OD600nm -Final OD600nm/initial OD600nm] x 100.

Estimation of water soluble and water insoluble glucans
The standard phenol/sulfuric acid estimation method was performed to quantify the water soluble and water insoluble glucans as described earlier (Yano et al. 2020). Brie y, S. mutans was cultured in TSB (supplemented with 1 % sucrose) in the presence and absence of 3, 5-DTBP and incubated anaerobically for 24 h at 37° C. After incubation, the cell free culture supernatant (CFCS) was harvested by centrifugation. The supernatant was subjected to ethanol precipitation to obtain extracellular water soluble glucans. The remaining cell pellet was resuspended in 1M NaOH and centrifuged to remove the bacterial cell and, the water insoluble (alkali-soluble) glucan was recovered by ethanol precipitation of the harvested supernatant.

Glycolysis pH drop assay
The level of the glycolysis pH drop of S.mutans was measured as earlier described method (Ban et al. 2012). The cells of S.mutans from the suspension cultures were centrifuged at 15000 g, washed once with salt solution (50 mM KCl and 1 mM MgCl 2 ) and resuspended in a salt solution with the presence and absence of 3, 5-DTBP (100µg/ml). The pH was adjusted to 7.2 to 7.4 with 0.2 M KOH solution. Glucose was then added to obtain a concentration of 1% (w/v) and the decrease in pH was assessed over a period of 60 min.

Terminal pH determination assay
To analyze the effect of 3, 5-DTBP on acid production, terminate pH assay was performed. Brie y, an overnight culture of S.mutans was inoculated with TSB (pH 7.4), with presence and absence of 3, 5-DTBP and incubated at 37° C for 24 hours. The pH of bacterial broth was determined at the before and after 24 h incubation. The changes in pH were recorded (Korithoski et al. 2007).

Lactic acid measurement
Brie y, the cells of S. mutans were harvested, washed twice with PBS and resuspended to a nal concentration of 1 x 10 8 CFU/ml in a 24 well plate with 1.5 ml buffered peptone water (BPW) supplemented with 0.2 % sucrose containing BIC of 3, 5-DTBP in each well and incubated for 120 min at 37° C. After incubation, planktonic cells were removed by centrifugation (8,000g, 5 min, 4° C), the supernatant (50 µl) was added to 2 ml of 0.2 % iron (III) chloride, stirred and absorbance was measured at 390 nm (Borshchevskaya et al. 2016).

Lactate dehydrogenase assay
To evaluate the lactate dehydrogenase assay (LDH) activity, the overnight bacterial culture (OD 600 = 0.8) was incubated at 37° C with Tris -HCl buffer, pH 7.0 containing 0.5 mg/ml of lysozyme for 1 h. After the incubation, the cell free supernatant (CFS) was collected from centrifugation at 10000g for 20 min at 4°C . Furthermore, the CFS was dialyzed with 10 mM phoaphate buffer saline (PBS) (pH 7.0) at 4° C for overnight. The dialyzed crude LDH protein content was measured by the Bradford method (Bradford 1976). After that, the LDH activity was determined by the previous method described by (Crow and Pritchard 1977). The crude LDH was pretreated with 3, 5-DTBP (at 100 µg/ml) at 30 min at room temperature, and the LDH activity was estimated by measuring the rate of nicotinamide adenine dinucleotide (NADH) oxidation at 340 nm. The standard reaction mixture (for 200 µg/ml) contained 180 µl of 50 mM PBS pH 7.0 with 0.167 mM NADH and 10 mM sodium pyruvate; 10 µl of 1 mM fructose 1, 6diphosphate (FDP) and 10 µl of pretreated LDH. The results were expressed as enzymatic activity compared to that of the control.

Enolase activity assay
To determine the enolase activity with permeabilized cells of S.mutans were measured by a method described earlier (Belli et al. 1995). Permeabilized cells were pretreated with 100 µg/ml concentration of 3, 5-DTBP for 30 min at room temperature. The enolase activity was observed by the formation of Acid tolerance assay Acid tolerance ability of S. mutans in the presence and absence of 3, 5-DTBP, was assessed by acid tolerance assay. S. mutans was grown till mid logarithmic phase and pelleted by centrifugation. The pellet was divided into two aliquots, one aliquot was directly resuspended in TSB at a killing pH of 3.5 and incubated at 37° C for 2 h. These cells were considered as unadapted cells. Then, the other aliquot was rst suspended in TSB at a pH of 5.5 and incubated at 37° C for 1 h to ease adaptation. The resulting adapted cells were then transferred to TSB at a lethal pH of 3.5 and incubated for 2 h at 37° C.
Cell fractions were removed from these unadapted and adapted cultures at regular intervals (at 0, 1 and 2 h) and their viability was tested by TSB agar plates at favorable pH (pH 7.5) for 48 h at 37°C (Viszwapriya et al. 2017).
The F 1 F 0 -ATPase activity was determined by using permeabilized cells of S.mutans by adding the cells to 10 % toluene followed by series of freezing and thawing as described earlier method. The F 1 F 0 -ATPase activity was evaluated by the release of inorganic phosphate in the following reaction mixture 75mM of Tris -maleate buffer (pH 7.0) containing 5mM adenosine -5 -triphosphate (ATP), 10mM of M g Cl 2, permeabilized cells along with BIC concentration of 3, 5-DTBP. The release phosphate was determined as previously described method (Bencini et al. 1983).
Auto aggregation assay S.mutans was grown in the presence and absence of 3, 5-DTBP (100 µg/ml) and incubated at 37° C for 24 h. The bacterial suspensions were then centrifuged and the pellet was resuspended in PBS (pH 7.4).
The suspensions were kept undisturbed and visually observed for aggregation at the interval of 10 min to

Growth curve and viability of S. mutans
To evaluate the effect of 3, 5-DTBP on growth of S. mutans, 100 ml of TSB was inoculated with 1 % of overnight culture of S. mutans in the presence and absence of BIC of 3, 5-DTBP. The zero hour OD was measured at 600 nm and the culture was incubated at 37° C. The OD values were taken at 3 h interval for 24 h and the growth curve was plotted as OD against time interval (Subramenium et al. 2015).
Further, the cell viability of S. mutans quanti ed using the XTT (2, 3-bis (2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay. Brie y, the XTT sodium salt and menadione were dissolved in sterile PBS (1 mg/ml) and acetone (1mM) respectively. For each experiment, XTT -menadione solution was prepared freshly at the ratio of 12.5.1 respectively. After incubation, both bio lm and planktonic cells were collectively removed from the 3, 5-DTBP treated and untreated wells. The cells were washed twice with sterile PBS and resuspended in the 175 µl PBS with 25 µl of XTT-menadione solution. This mixture was incubated in dark at 37° C for 8 h. The viability of tested strains was measured spectrophotometrically by the reduction of XTT-menadione into the orange colored formation at 490 nm (Vijayakumar et al. 2020b).

RNA extraction and cDNA preparation
The total RNA of S. mutans grown in the presence and absence of 3, 5-DTBP for 24 h was isolated using a method described earlier (Oh and So 2003). The isolated RNA samples were reversed transcribed using High capacity cDNA reverse transcription kit (Applied Biosystems) as per the manufacturer's instructions. Then cDNA samples were used for subsequent real-time polymerase chain reaction (qRT-PCR) analysis.

Real-Time PCR (qRT-PCR) analysis
The qRT-PCR was performed using 7500 thermal cycler system (Applied Biosystems, USA) with the SYBR Green chemistry kit (Applied Biosystems, USA) following the manufacturer's instructions. The oligonucleotide primers were designed with Primer3 software and are listed in Table. 1. The cycling parameters were as follows: initial denaturation of 5 min at 95° C, followed by 40 cycles of denaturation at 95° C for 30 s, annealing at 56° C for 30 s, extension at 72° C for 30 s and a nal extension at 72° C for 1 min. The data was acquired at each cyclic extension step. All the experiments were performed and analyzed in triplicate. The 16S rRNA was used as calibrator to normalize the gene expression. The relative expression levels of target genes were calculated using the 2 -ΔΔC t method (Livak and Schmittgen 2001).

Statistics
All the experiments were performed in triplicate at least three times. The data were expressed as mean ± standard deviation (SD). One way ANOVA, 5-DTBP was performed using SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). Results with *P <0.05 and was indicated statistically signi cant.

Results
Screening of phytochemicals antibio lm activity against S. mutans Antibio lm potential of ve different phytochemicals (100 µg/ml) was assessed by 24 well MTP assay along with CV staining. The results obtained from, OD at 570 nm was signi cantly reduced upon treatment with 3, 5-DTBP (p<0.05) whereas remaining compounds slightly inhibited the bio lm formation of S. mutans and found to be no statistical signi cant (Fig. 1a) Determination of bio lm inhibitory concentration (BIC) of 3, 5-DTBP S. mutans bio lm was treated with various concentrations of 3, 5-DTBP ranging from 20 -120 µg/ml. Concentration dependent bio lm inhibition was observed upon 3, 5-DTBP treatment with maximum inhibition (91 %) was observed at 100 µg/ml concentration (p<0.05), the above concentrations do not show any signi cant effect hence this concentration was xed at BIC of 3, 5-DTBP and further experiment was done with this concentration (Fig. 1b).

Microscopic analysis
Antibio lm potential of 3, 5-DTBP was con rmed by light microscopic and CLSM analysis. Light microscopic images showed signi cant reduction in bio lm upon 3, 5-DTBP treatment in S. mutans. In control slide shows a dense bio lm formation covered on the entire surface wherein minimum number of cells was observed in the surface treated with BIC of 3, 5-DTBP (Fig. 1c). Further, bio lm architecture arrangements were assessed by CLSM and the results con rmed the antibio lm activity of 3, 5-DTBP. BIC of 3, 5-DTBP treated slides showed a signi cant reduction of bio lm matrix thickness and minimum number of cells adhered on to the surface compared to the control (Fig. 1d).
Effect of 3, 5-DTBP on adherence of S. mutans Adherence ability plays a crucial role in oral microorganisms; in particular, S. mutans has able to attach the tooth surface with the support of sucrose dependent and sucrose independent progress. The present study showed that 3, 5-DTBP at BIC signi cantly reduced the sucrose dependent and sucrose independent adherence activity by 65 % (p<0.05) and 58 % (p<0.05) respectively (Fig. 2a). The present nding indicated that the 3, 5-DTBP treatment is more e cient to inhibit sucrose dependent adherence wherein compared than sucrose independent adherence.

Effect of 3, 5-DTBP on CSH of S. mutans
Bacterial cell surface hydrophobicity is an important virulence factor responsible for bacterial adherence. The MATH assay results showed that 3, 5-DTBP (100 µg/ml) treatment greatly reduced the CSH activity by 77 % when compared to the untreated control. The CSH inhibition at BIC was found to be no statistical signi cant (Fig. 2b).

Inhibition of water soluble and insoluble glucans
The obtained results showed that 3, 5-DTBP treatment greatly reduced the water soluble and water insoluble glucans production by 48 % and 87 % wherein compare than respective controls (Fig. 2c). The decreased level of water insoluble glucan synthesis at BIC was found to be statistically signi cant (p<0.05) and water insoluble glucan inhibition was found to be no statistical signi cant.

3, 5-DTBP inhibits acidogenicity of S. mutans
The present studies observed the effect of 3, 5-DTBP on acidogenicity of S. mutans monitoring by glycolytic pH drop assay, terminal pH, lactic acid quanti cation assay and LDH assay. The results showed that the 3, 5-DTBP treatment at BIC greatly reduced the initial rate of S. mutans cells pH drop. In control sample nal pH value was recorded at 4.6 whereas the 3, 5-DTBP treated cells nal pH value was recorded at 6.7 respectively (Fig. 3a). The terminal pH assay results showed that the 3, 5-DTBP treated cells terminal pH was recorded at 5.5 whereas the control sample terminal pH was recorded at 4.4 respectively ( Table 2). Also, the 3, 5-DTBP treatments signi cantly reduced the S. mutans lactic acid production (p<0.05) (Fig. 3b) and LDH activity by 80 % (p<0.05) respectively (Fig. 3c). As well as 3, 5-DTBP treatment greatly reduced the enolase activity by 70 % respectively. The enolase inhibition at BIC was found to be no statistical signi cant (Fig. 3d), which were consistent with the results of glycolytic pH drop assay.

Auto-aggregation assay
Aggregation is an important process; the bacterial cells are fused and settled on the bottom and construct the microcolonization. The auto-aggregation process is essential for construct aggressive bio lm formation. Then, the effect of 3, 5-DTBP on the auto-aggregation activity of S. mutans was assessed. The results showed that 3, 5-DTBP treated cells aggregation rates are faster than the control cells. This property of 3, 5-DTBP initiates to inhibit the bio lm formation by the new strategy. The treated cells showed 85 % auto-aggregation, even control cells showed 41 % of auto-aggregation, when 30 min of incubation in undisturbed conditions (Fig. 5a). The auto-aggregation reduction at BIC was found to be no statistical signi cant.
Effect of 3, 5-DTBP on H 2 O 2 sensitivity of S. mutans H 2 O 2 sensitivity assay was performed to test the ability of 3, 5-DTBP to sensitize S. mutans cells towards oxidative stress. The results showed that 3, 5-DTBP signi cantly reduced the number of cells resistant to H 2 O 2 when compared to control cells (Fig. 5b). The oxidative stress reduction at BIC was found to be no statistical signi cant.

Effect on 3, 5-DTBP on growth and cell viability of S. mutans
The present study con rms the non-bactericidal property of 3, 5-DTBP against S. mutans, growth curve analysis was done in the presence and absence of 3, 5-DTBP (100 µg/ml). Results from growth curve assay, it is clear that 3, 5-DTBP do not affect the S. mutans growth pattern wherein compare than control (Fig. 6a). Further, the non-lethal effect of 3, 5-DTBP was also assessed by using metabolically active cells grown in the presence and absence of 3, 5-DTBP. The results showed no signi cant variance between the 3, 5-DTBP treated and non-treated metabolic cells. (Fig. 6b). These obtained results at BIC were found to be no statistical signi cant.

Discussion
The present study investigates the anticariogenic potential of 3, 5-DTBP against S. mutans which has been considered as an etiological pathogen to cause dental caries. 3, 5-DTBP exhibited a concentration dependent antibio lm (100 µg/ml) e cacy against S. mutans (Fig. 1b). Hence, 100 µg/ml concentration was considered as BIC of 3, 5-DTBP. Light and CLSM microscopic analysis to con rm the concentration dependent antibio lm e cacy of 3, 5-DTBP against S. mutans (Fig. 1c, d). Furthermore, 3, 5-DTBP non-bactericidal property was con rmed by growth curve assay and XTT assay (Fig. 6a, b). The present results were similar to the previous report (Rathna et al. 2016) who has reported that 3, 5-DTBP isolated from Pleurotus orida inhibits C. albicans bio lm formation without affect fungal growth.
The bacterial bio lms are made up of polysaccharides, proteins, lipids, extracellular DNA (eDNA), lipoproteins and lipooligosaccharides (Flemming and Wingender 2010). Among these extracellular polysaccharides (EPS) was found to be an abundant component in the bio lm matrix (Koo et al. 2010). In S. mutans EPS is synthesized by glucosyltransferases (gtf), gtfB produces water insoluble glucans, which found the scaffold of the EPS matrix and gtfC produces the water insoluble and soluble glucans, which is responsible for providing the binding site and structure of S. mutans bio lms (Xiao et al. 2012). The present study, 3, 5-DTBP at 100 µg/ml concentration exhibited a signi cant inhibition in the synthesis of water soluble and water insoluble glucans (Fig. 2c). The phenomenal reduction of adherence and EPS production in 3, 5-DTBP treatment, which signi es the anti-adherence mediated bio lm inhibition pro ciency.
Adhesion, acidogenicity (ability to synthesis acid) and aciduricity (ability to tolerate acid) are the major virulence factors in S. mutans (Banas 2013). Hence, the present study mainly focused on these virulence productions. Adherence plays a crucial role in oral microorganisms, in particular, S. mutans attaching the tooth surface, which leads to cascade of bio lm formation (Matsumoto et al. 1999). Generally, S. mutans adherence was occurred in two distinct mechanisms such as sucrose dependent and sucrose independent mechanisms. Several studies are documented that sucrose dependent adherence plays a crucial role in causing dental caries and bio lm formation compared to sucrose independent adherence. Here, 3, 5-DTBP signi cantly inhibited both sucrose dependent adherence and sucrose independent adherence by 65 % (p<0.05) and 58 % (p<0.05) respectively (Fig. 2a). The present nding was compared with the previous study (Gowrishankar et al. 2014) who has reported that Bacillus amyloliquefaciens (MMS-50) bioactive metabolite cyclo (L-leucyl-L-prolyl) inhibits sucrose dependent and sucrose independent adherence by 50 % and 55 % respectively. Cell surface hydrophobicity mainly involved host -pathogen interactions and bacterial cell attachment on tooth surface via hydrophobic interactions. The previous studies reported that restrict of S. mutans hydrophobic nature, which inhibiting the bio lm formation and reduces the opportunities of bacterial attachment and caries development (Khan et al. 2012). The CSH assay revealed that 3, 5-DTBP has the potential to modify the cell surface hydrophobic properties of S. mutans (Fig. 2c).
Acidogenicity is an important virulence factor, which is responsible to produce high pH acid from the dietary carbohydrate (glucose) with the help of glycolysis pathway. In this progress, several enzymes are involved directly to help us synthesis the acid. Lactate dehydrogenase is an important enzyme mainly involved in the conversion of pyruvate into lactate acid in anaerobic condition. As well as the lack of LDH, affect the NAD+ conversion from NADH. NAD+ is required when glyceraldehyde-3-phosphate dehydrogenase oxidizes glyceraldehyde-3-phosphate in glycolysis. Moreover, the increasing level of NADH affects the redox potential of the cell, which is documented to be toxic for S. mutans (Postma et al. 1993). This fact has driven the study to evaluate the LDH activity of S. mutans in the presence of 3, 5-DTBP. The results showed the signi cant reduction of LDH production compared to control sample (Fig.  3c). Moreover, the present study found that 3, 5-DTBP treatment signi cantly inhibited the enolase activity (Fig. 3d). Enolase is a glycolytic enzyme responsible for the formation of phosphoenolpyruvate, in bacteria utilized phosphoenolpyruvate as the source of energy through the phosphotransferase system. Then, the lack of enolase activity could be reduced phosphoenolpyruvate production which leads to decrease the acidogenesis (Xu et al. 2011). The reduction of acidogenesis ability was further assessed by glycolyic pH drop assay. The results unveiled that 3, 5-DTBP treatment signi cantly reduced the initial and nal rate of pH drop signifying the inhibition of acid production ability of S. mutans (Fig. 3a), the lack of enzymatic production could be a possible reason to diminish the bacterial glycolytic pathway. In addition, terminate pH assay to con rm the impairment of acidogenic property, the results showed that 3, 5-DTBP treated cells signi cantly maintain the pH level compared to control (Table 2). Furthermore, the glycolytic pH drop assay result implies impairment of S. mutans acid tolerance ability upon 3, 5-DTBP treatment.
F 1 F 0 -ATPase plays a crucial role in S. mutans acid tolerance ability; it regulates the bacterial cell pH gradient across the membrane. F 1 F 0 -ATPase regulates cytoplasmic alkalization because alkaline pH is essential for regular cell functioning and enzymes activity (Hamilton and Buckley 1991). The previous study suggests that impairment of F 1 F 0 -ATPase enzymatic activity affects the acid tolerance ability ). Hence, the insu ciency of F 1 F 0 -ATPase may be involved in increasing the cytoplasmic acidity and subsequently decreased acid tolerance. The present study revealed that 3, 5-DTBP treatment greatly affects the F 1 F 0 -ATPase activity (Fig. 4). Further, the acid tolerance assay to con rm, 3, 5-DTBP treated cells lost their ability to survive on the acidic conditions (Table 3). Hence, the present study con rms the inhibition of S. mutans cariogenic property (adherence, acidogenesis and acid tolerance) by targeting the biomolecular mechanisms responsible for the S. mutans cariogenesis.
In addition, 3, 5-DTBP anticariogenic potential was con rm by transcriptomic analysis. The results showed the downregulation of gtf genes (gtfB, gtfC and gtfD). It could be a positive sign to eradicate the S. mutans virulence production. Gtfs plays a crucial role in synthesis of glucans, which is essential for adherence on tooth surface. Generally, gtf enzymatic action was regulated by gtfC, gtfD, gtfB genes. The gtfB and gtfC genes are working in a similar model and synthesis the end product of water insoluble glucans. On the other way, gtfD gene synthesis water soluble glucans (Yamashita et al. 1999). Hence, the downregulation of gtf genes might be a possible reason to impedes the glucans production upon 3, 5-DTBP treatment (Fig. 7). The present result was compared to previous study (Ren et al. 2016) who has documented that interfering of glucosyltransferase could reduce the ability of bio lm formation and cariogenic virulence of S. mutans. Glucans binding protein plays an important role to regulate the a nity of glucans which increasing the bacterial adherence rate to the tooth surface. The results showed the signi cant downregulation of gbpB could responsible for reduction of bacterial adherence upon 3, 5-DTBP treatment (Fig. 7).
The two component signal transduction systems (TCSTS) which responsible to regulate bacterial bio lm formation, adaptation, survival and virulence production. These signal transduction systems act as molecular switches, which modify the gene function to survive in the extreme environment (Senadheera et al. 2005). The TCSTS is accomplished by two regulatory elements vicK (histidine kinase) and vicR (response regulator). The previous study reports that vicR gene controls the bio lm formation and gtfBCD genes expression, though downregulation of vicR greatly reduced the bio lm formation and glucan synthesis (Senadheera et al. 2005). The present investigation found the vicR gene downregulation upon 3, 5-DTBP treatment; it could be a possible reason for inhibition of bio lm formation and suppression of gtf expression. The result was parallel to the previous study betulin inhibits S. mutans bio lm formation via impedes of vicR (Viszwapriya et al. 2017) (Fig. 7). Moreover, the current study also found that 3, 5-DTBP treatment modi es the growth nature of S. mutans in a liquid medium. The obtained result showed the overnight culture of control cell suspension is evenly formed, in contrast, 3, 5-DTBP treated cells are co-aggregate together and settled at the bottom of the medium (Fig. 5a). The previous study has reported a similar form of aggregation observed in vicK mutans strains (Senadheera et al. 2005). VicRK genes are arranged in an operon model and their expressions are co-regulated (Tremblay et al. 2009). Then, the downregulation of vicR could downregulates the vicK expression, which could be a possible reason behind the cell aggregation upon in 3, 5-DTBP treatment. In addition, vicK mutant strains formed unstable bio lms. The present ndings added one more attention, as 3, 5-DTBP treatment correspondingly inhibits bio lm formation in the existence of sucrose.
relA gene encodes guanosine tetra-phosphate which is responsible to regulate acid tolerance (Liu et al. 2011). The downregulation of relA gene could be a possible reason for impairment of acid and stress tolerance. comDE gene regulates the S. mutans quorum sensing mechanism and mediated virulence productions (Senadheera and Cvitkovitch 2008). Hence, the suppression of comDE gene expression directly affects quorum sensing as well as mediated bio lm formation, acid tolerance and acid production. In addition, smu0630 is a hypothetical protein involved in bio lm formation ). The downregulation of smu0630 (Fig. 7) gene attenuates the bio lm formation, which may is an alternative approach to reduce/control S. mutans bio lm formation (Liu et al. 2011).
In the present study demonstrate the antibio lm potential of 3, 5-DTBP explored antibio lm and anticaries activity against S. mutans. 3, 5-DTBP at 100 µg/ml inhibited the bio lm formation and signi cantly reduced the adhesion, acidogenesis and aciduricity property through impairment of enzymatic productions and downregulating the gtf and vicR/K virulence genes. Based on this data, we believed that 3, 5-DTBP as a potential antibio lm and anticaries agent could control the S. mutans cariogenesis.
Declarations 52. Zhang Z, Liu Y, Lu M, Lyu X, Gong T, Tang B, Wang L, Zeng J, Li Y (2020) Rhodiola rosea extract inhibits the bio lm formation and the expression of virulence genes of cariogenic oral pathogen Streptococcus mutans. Arch Oral Biol 19:104762.