Antibodies against dextransucrase from Streptococcus mutans display anti-biolm and growth suppressing activities

Dextransucrase produced by Streptococcus mutans play an essential role in the formation of dental caries by synthesizing exopolysaccharides from sucrose, an important metabolite of the organism. In this study we report the location of dextransucrase in Streptococcus mutans cells and describe that antibodies raised against dextransucrase inhibited biolm formation and reduced the adherence and hydrophobic properties of Streptococcus mutans. Western blot analysis and immunoelectron microscopy revealed that dextransucrase is located abundantly in the membrane fraction in S. mutans cells. Scanning electron microscopy and uorescence microscopy revealed reduced cell density, impaired bioim (plaque) formation in presence of dextransucrase antibodies. Genes associated with bioim formation in S. mutans such as GtfB, GtfC, BrpA, relA, Smu630, vicK were down regulated (50–97%) in presence of the enzyme antibody. Presence of enzyme antibodies reduced adherence of S. mutans cells to glass surfaces by 58% and hydrophobicity by 55.2%. However dextransucrase antibodies did not affect acid production by S. mutans, under the experimental conditions. Immunohistochemistry studies with certain human samples displayed no cross reactivity with dextransucrase antibody. These ndings suggest that antibodies against dextransucrase exhibit a profound inhibitory effect on the vital cariogenic factors of S. mutans and have no cross reactivity with human tissues tested, thus implying that dextransucrase could be a promising antigen to study its anticariogenic potential.


Introduction
Dextransucrase (EC 2.4.1.5) produced by Streptococcus mutans is primarily an extracellular enzyme but its exact location in S. mutans cells is unknown. It synthesizes mainly α-(1-3) rich and α-(1-6) dextran polysaccharides from its exclusive substrate sucrose 1 which help in the attachment of microbes to the tooth surface in the form of plaque and bio lm formation, causing the infection 2 . The attachment of microbes onto the tooth surface is the result of hydrophobic interactions between complementary molecules on the microbe and the tooth surface 3 . Various factors associated with the establishment of caries include adhesion, bio lm formation, acidogenicity and aciduricity. S. mutans has a remarkable ability to tolerate acidic environment 4 and the acids produced by the metabolism of dietary carbohydrates lead to the dissolution of tooth enamel and subsequent decalci cation, cavity formation and hence degradation of calci ed dental tissue 5 . The adaptation to high acidic environment is followed by changes in the expression of different genes and proteins associated with protein transport, nuclear acid metabolism, signal transduction, energy metabolism which includes overexpression of 169 genes and repression of 108 genes 6 . The enzymes and proteins whose expression gets changed at acidic pH mostly belongs to stress proteins, amino acid synthesis, acid production, glycolysis pathways and membrane proteins. The adhesion is also facilitated by adhering to the pre-adhered bacteria via lectin links which helps in bio im synthesis leading to the plaque formation 7 . Adhesion of bacteria on the oral surfaces is also promoted by various surface proteins which includes antigen B, antigen I / II, IF, SpaP, P1, and SR. Gtf genes like GtfB, GtfC, and Gtf D also appears to be essential for the cariogenesis. Gtf B forms water-insoluble α-1,3 binding glucans, GtfC produces α-1,3 and α-1,6 binding glucans and GtfD produces water soluble α-1,6 binding glucans or dextran, which are required for the bio lm mediated plaque formation 8 , 9 . Glucan binding proteins synthesized by S. mutans helps in the maintenance and architecture of bio lms formed by other oral organisms also 8 . The ability of S. mutans to form bio lms is the potential factor for the development of caries formation 10 .
Several studies regarding immunotherapy for caries based on both in vivo and in vitro studies have been explored 11 . Antibody induction by active immunization with S. mutans antigens has been reported to be an effective method to provide protection against experimental dental caries. Other adhesion molecules of S. mutans, which have been used as antigens for vaccine development include AgI/II, GTFs, GBPs and Pst system (phosphate-binding-protein) 12 . The antibodies elicited by these antigenic molecules target the speci c virulence factors of the pathogens and hence minimises the development of caries formation.
The subunit vaccines containing AgI/II or GBP or GTFs from S. mutans 13 , recombinant vaccines prepared from the genome targeting the expression of speci c antigen of the pathogen 14 have also been reported.
However antibodies against these molecules are reported to have cross reactivity against skeleton muscles and heart tissues 15 , but antibodies against dextransucrase when tested for cross reactivity with human heart tissues and various mammalian tissues revealed negative results 16 .
The direct relationship between dextransucrase activity and cariogenicity makes it a good candidate to study its anticariogenic potential. Thus in the present study we evaluated the localization of dextransucrase in S. mutans cells and the effect of anti-dextransucrase antibodies on the bio im formation and other associated cariogenic factors. These results are reported herein.

Results
Dextransucrase Localization in S. mutans cells.
The site of location of dextransucrase in S. mutans was determined by measuring dextransucrase activity in different fractions of S. mutans cells which was further validated by immunoelectron microscopy and western blot analysis.
Cell pellet obtained after centrifugation of 24 hours. culture of S. mutans was lysed by sonication which was subjected to differential ultracentrifugation. The pellets obtained at 2000g, 8000g, 40000g and 100000g centrifugation contained undisrupted cells, cell wall fraction, cell membrane fraction and cytoplasmic fraction respectively 24 . The pellets were suspended in 20mM sodium maleate buffer pH 6.8 and each fraction was analysed for dextransuccrase activity. Dextransuccrase activity in 40000g fraction was high as compared to other cell fractions indicating that dextransucrase is largely associated with the membrane fraction of the S. mutans cells.
Immunoelectron microscopic location of dextransucrase in S. mutans cells Using the antibody raised against dextransucrase as well as gold-labeled IgG, dextransucrase antigen was localised by detecting the immune complexes formed by dextransuccrase antibody and the dextransucrase present in the S. mutans cells. As shown in Fig. 1 the immuno-gold labelling was observed more on the surface of the cells as compared to the cytosol, which demonstrated that dextransucrase is largely associated with the membrane part of S. mutans cells. This was apparent even in the cells undergoing cell division. The immune-gold particles seen outside the cells presumably represent the excretory enzyme in the medium.
Reactivity of S. mutans fractions with dextransucrase antibody.
The immunoblot analysis (Fig. 2a) revealed that reactivity was more pronounced in membrane fractions of S. mutans as compared to the cytosolic fractions, this further corroborated that dextransuccrase is largely associated with the membrane fraction of the cells. Densitometric scan of the immunoblot carried out by ImageJ software is shown in Fig. 2b.
Dextransucrase antibody inhibited bio im formation by S. mutans in vitro.
S. mutans were allowed to form bio lms for 20h at 37 o C on glass cover slips that were deposited in 6well culture plates in BHI media supplemented with 5% sucrose. S.mutans cells grown on cover slips were xed with 2% glutaraldehyde and 4% formaldehyde and sputter coated with platinum in carbon. Images were taken from magni cations 500X, 1000X, 3000X and 6000X. As shown in Fig. 3  The effect of dextransucrase antibody on bio im formation was also studied using propidium iodide (PI) staining by uorescence microscopy. S. mutans cells grown on saliva coated glass coverslips in presence and absence of dextransucrase antibody were xed and stained with propidium iodide solution (1µg/ml) as mentioned in materials and methods. These results illustrated that dextransucrase antibodies reduced the bio im formation by S. mutans. The cells in the control samples showed clumping of cells in the polysaccharide matrix however the cells grown in presence of the antibody were well dispersed, suggesting inhibitory effect of antibody on bio im formation by S. mutans (Fig. 4).
Dextransucrase antibodies down regulated the expression of bio lm forming genes in S. mutans Transcriptional expression of bio lm forming genes GtfB, GtfC, BrpA, Smu630, RelA and VicK was determined by using Real-Time PCR system. The speci c primers and reaction conditions are described in Table 1 of material and methods section.16sRNA was used as an invariant internal control. The data of quantitative analysis is represented as ΔCT (ΔCT = CT target -CT 16sRNA) and 2 -∆∆CT method was used for the determination of fold change in mRNA expression [25].
Effect of dextransucrase antibodies on adherence of S. mutans to glass surfaces Adhesion of bacterial cells on the tooth surface is the prerequisite for the bio im formation which is initiated by attachment of cells to the surfaces followed by assembly of cells leading to plaque formation. The effect of dextransucrase antibody on the adherence of S. mutans cells on the glass surfaces was studied by allowing the growth of bacteria in presence (Test) and absence (Control) of dextransucrase antibody in glass tubes kept at 30° containing BHI medium with 5% (w/v) sucrose at 37°C for 24 h as mentioned in methods section. The effects of dextransucrase antibodies (30µg) on the adherence of S.mutans is shown in Fig. 6(a). Results with dextransucrase antibody exhibited 58.3% reduction in the adherence of S. mutans suggesting considerable inhibitory effect on the adherence of the organism.

Dextransucrase antibodies decreased cell surface hydrophobicity of S. mutans
The effect of dextransucrase antibody on the hydrophobicity of S. mutans grown in presence of 30 µg of antibody in BHI medium supplemented with 5% sucrose for 20 hours at 37°C was carried out. The samples without antibody were taken as the controls. These results are shown in Fig. 6(b). A considerable reduction in the hydrophobicity of S. mutans was observed under these conditions. The hydrophobicity index was 55.2% which was less than the standard value of hydrophobicity index.
Effect of antibodies on acid production by S. mutans Acid production by S. mutans was analysed by culturing the bacteria in presence (Test) and absence (Control) of dextransucrase antibody at 37°C. Culture media without inoculum was taken as control. The pH of the culture was measured at 0 h and at 24 h. As shown in Fig. 6(c) there was essentially no effect of dextransucrase antibodies on the acid production by S. mutans, under the experimental conditions. The data indicated that dextransucrase antibodies did not affect the acid production by S. mutans.

Cross reactivity of dextransucrase antibody with human samples
The antibodies raised against dextransucrase were tested for the reactivity with different human tissues by performing immunohistochemistry. The positive control was prepared from the heart muscle tissue which was immersed in the antigenic protein and xed before preparing the slides. As shown in Fig. 7 antibodies raised against dextransuccrase did not show any reactivity with any of the tissues tested, which further demonstrated that there was no cross reactivity with the human tissues tested.

Chemicals
Analytical grade chemicals were used in this study. Brain heart infusion broth (BHI), Agar, peptone, sucrose, exogenous dextrose, glucose, maleic acid, NaCl and ethanol were obtained from HiMedia Pvt.
Puri cation of dextransucrase from S. mutans and production of antibodies.
Dextransucrase was puri ed to homogeneity from the culture supernatant of S. mutans MTCC-890 grown at 37 °C for 36 h by 55% ammonium sulphate precipitation followed by gel ltration chromatography using Sephadex G-200 and treatment with PEG-400 (polyethylene glycol 400) as reported earlier 16 .
Polyclonal antisera against dextransucrase puri ed from S. mutans was raised in New Zealand White rabbits immunised subcutaneously with puri ed dextransucrase (500μg) mixed with complete Freund's Complete Adjuvant (F5881, Sigma, USA). Serum was collected after two weeks of last booster and analysed for dextransucrase speci c antibody by Dot blot assay, confocal microscopy, ELISA and western blot analysis as described previously 16 . Serum IgG was puri ed by a nity chromatography using Protein A Sepharose Column (Bio Vision, USA).

Cell fractionation of S. mutans
Cell fractionation was carried out by separating the cells from culture media by centrifugation. The cell pellet was treated with lysozyme 0.25mg/ml in 50mM sodium maleate buffer pH 6.8 for half an hour in ice and then kept in a water bath at 37°C for 5 min. The cells were lysed by sonication using cell sonicator (Sonics-Vibra VCX-750 USA) for 10 min and the pulse rate was 10 sec on and 10 sec off. After cell disruption the sample preparation was subjected to differential centrifugation using ultracentrifuge Beckman coulter (Optima XPN-100). The sample preparation was centrifuged at 2000g to separate unbroken cells followed by 8000g for 10 min, 40000g for 30 min and 150000g for one hour to collect different fractions of S. mutans cells. All the procedures were carried out at 4°C. The pellets obtained at each step were suspended in 20mM sodium maleate buffer pH 6.8 and stored at -20°C and analysed for dextransucrase activity and a nity for dextransucrase antibody.

Immunoelectron Microscopy
Immunoelectron microscopy was carried out for detecting intracellular and surface location of dextransucrase utilizing antigen-antibody reactions 17 . The cells were xed in 0.2% glutaraldehyde and 4% paraformaldehyde in PBS for 2-hours at room temperature. The xed cells were dehydrated in ascending grade of ethanol (20%, 50%, 70%, 90%, and 100%) and embedded in LR white resin (Hard grade) (TAAB, UK) at 55°C. Thin sections of 80-90 nm thickness were cut using ultra microtome and mounted onto 300 mesh nickel grids. The grids were rst incubated in 1% cold-water sh gelatin made in 0.01M phosphate buffer saline (PBS) for 30 minutes to block nonspeci c sites followed by incubation in primary antibody (Dextransuccrase antibody) diluted in the ratio of 1:200 overnight at 4°C. The grids were washed three times one minute each in diluent buffer and incubated in secondary antibody (goat antirabbit) conjugated with 10 nm colloidal gold (dilution: 1: 50; TAAB, UK) for 2 hours at room temperature followed by washing three times with buffer and water one minute each. The grids were stained for ve minutes with uranyl acetate followed by brief washing with distilled water. The grids were blotted dry and observed at a magni cation 29000X at 200kV under transmission electron microscope Tecnai G20 (FEI Company, The Netherlands).

Scanning Electron microscopy (SEM)
Scanning electron microscopy was performed following the method described by Ansari et al. 18 . Cultures of S.mutans with OD 0.01were grown on glass coverslips immersed at the bottom of the 6-well culture plates to allow the formation of bio lms. Puri ed IgG was added to the wells and wells without addition of IgG served as the control. After 36 hours of incubation at 37 o C the glass coverslips were carefully removed from the wells and washed three to four times with PBS. The cells were xed by 4% formaldehyde and 2% glutaraldehyde in PBS pH 7.4 for 2 hours followed by 3-4 times PBS washings. After xation the glass coverslips were dehydrated by suspending in different percentages of ethanol (10-100%) for 15min each and dried using critical point dryer CPD (EMS 850 JEOL Japan). Sample specimens were sputter coated with platinum in argon gas using sputter coater (JEC-3000FC JEOL Japan) and observed under SEM (JSM-IT300 JEOL Japan). The images of treated cells and control were compared.

Fluorescence microscopy
Cultures of S.mutans were grown on glass cover slips in 6-well culture plates with OD 0.01 to allow the formation of bio lms on the glass coverslips. Puri ed IgG fraction of dextransucrase antibody was added to the wells in triplicates labelled as treated and wells without addition of IgG served as control. After 24 hours of incubation at 37°C the glass coverslips were carefully removed from the wells and washed three to four times with PBS. The cells were xed in xation buffer containing 4% formaldehyde and 2% glutaraldehyde in PBS pH 7.4 for 2 hours followed by 3-4 times washings with PBS. The samples were stained with propidium iodide (1µg/ml) for one hour followed by washing with PBS and analysed under uorescence microscope (EVOS M7000 Imaging System Thermos Fisher Scienti c) as described by Wen et al. 19 .

Cell adherence
The effect of the dextransucrase antibodies on the adhesion of S. mutans to smooth glass surface was studied by the method of Hamada and Torii 20 . The organism was grown in a glass tube at an angle of 30° containing 10 ml of BHI medium with 5% (w/v) sucrose at 37°C for 24 h. The appropriate controls of BHI were run simultaneously. The cell adherence was quanti ed spectrophotometrically by measuring O.D. at 600 nm using untreated culture as the control. All the determinations were done in triplicates. Cell Surface hydrophobicity.
Cell surface hydrophobicity was determined by following the modi ed method of Martin et al. 21 . S. mutans MTCC 890 were inoculated to fresh BHI media and incubated for 12 hours in presence and absence of antibody (IgG). The bacterial cells were pelleted out and washed twice with sterile saline (0.85%) and optical density of the suspended cells in normal saline was adjusted to 0.3 at 600 nm. Thereafter 0.50 mL of toluene was added to 3.0 mL of the bacterial cell suspension in glass tubes which were agitated uniformly on a vortex mixer for 2 minutes and allowed to equilibrate for 10 minutes at room temperature. The absorbance at 600 nm of the aqueous phase separated from hydrocarbon phase was estimated using spectrophotometer. The hydrophobicity index, expressed as a percentage, was calculated as: [(OD initial -OD nal) / OD initial] × 100.

Acid production
The method of Ciardi et al. 22 was used to determine acid production by S. mutans. 5% (w/v) of sucrose in 5 ml of BHI broth containing serum antibody was added with 100 µl of 18 h cultures of S. mutans. The pH of the bacterial medium was assessed at 0 h and after 24 h of incubation. Except otherwise stated all determinations were done in triplicates and suitable controls were run simultaneously.

RNA extraction and cDNA Synthesis
Total RNA was extracted from the bacterial cells using TRizol (Sigma Aldrich USA) reagent according to manufacturer's instructions. RNA quanti cation and purity was determined by the Nano Drop ND-1000 spectrophotometer. RNA extraction was immediately proceeded for cDNA synthesis after quantitative assessment.
cDNA was synthesized from 1µg of RNA isolated from S. mutans cells using random hexamer primers and Revert Aid First Strand cDNA synthesis Kit (Thermo Scienti c) as per the user instructions in total reaction mixture of 20µl. The reaction mixture was incubated at 65°C for 5 minutes and then mixed with 5X reaction mix 4µl, RiboLock RNase inhibitor (20 Units/µl) 1µl 10mM dNTP mix 2µl, RevertAid M-MuLV RT (200 U/µl) 1µl. The reaction was incubated at 42°C for 60 minutes followed by 70°C for 10 minutes. The reaction was terminated and cDNA was stored at -20°C till used for qRT-PCR analysis.

qRT-PCR
Real time Polymerase Chain Reaction (RT-PCR) (relative quanti cation) was done using StepOne Plus Real-Time PCR system (Applied Biosystems, Life Technologies) by SYBER Green method. The relative expression of GtfB, GtfC, relA, brpA, smu630 and vicK was carried out using speci c primers as given in the Table 1 Immunohistochemical analysis was carried out following method of Malhotra et al. 23 to check the cross reactivity of dextransuccrase antibody with the mammalian tissues. Reference slides were prepared from the tissue samples of the rabbit and pre-prepared slides of human tissues were obtained from Department of Histopathology, PGIMER Chandigarh (India).

Discussion
Dental caries is a preventable bacterial infectious disease with multifactorial etiology. To control this disease an antimicrobial strategy will be appropriate to minimize the aciduric and acidogenic bacteria to reduce the demineralisation process. A number of efforts have been made to identify the antimicrobial agents and products for the reduction and removal of bacteria from tooth surfaces 26, 27 .
Dextransucrase is a secretory enzyme produced by S. mutans. It is present both in cell membrane and in supernatant fractions. The distribution of dextransucrase in the cell and supernatant fractions depends upon the presence of sucrose in the medium 28 . Cell associated dextransucrase is present either on the exterior surface of cell, which have an essential role in the cell adherence and is susceptible to proteolytic degradation 2 . In the present study localization of dextransucrase in the cells of S. mutans revealed that dextransucrase activity was present in all the pellet fractions of S. mutans cells obtained at 2000g, 8000g, 40000g and 100000g centrifugation, but was found mostly in the membrane fraction of the cells. Similar results were obtained by immunoelectron microscopic studies. The immunoblot analysis also showed more intense band in the membrane fraction as compared to the cytosolic fraction demonstrating that dextransucrase is largely associated with the membrane part than in the cytosolic region of S.mutans.
Bio lms formed by various pathogenic bacteria are involved in many diseases such as cystic brosis, endocarditis and many infections which arise from bio lms on arti cial valves, catheters 29 . Bio lm formation proceeds in multiple steps which involve pellicle formation which is a combination of mucin, glycoproteins and other proteins, followed by bacterial adhesion to the pellicle and maturation of the bio lms 30 . Since bio lms develops tolerance towards various antimicrobial agents, thus a potent agent against caries must prevent the pathogen from establishing bio lm formation. In this study results from SEM demonstrated that bio lm was disrupted showing scattered cells in treated samples. The cell density was reduced in the treated samples depicting disruption in the 3D structure of the bio lms as compared to control samples where the cells were clumped forming aggregates in the exopolysaccharide matrix with higher cell density. Further the uorescence microscopy data also corroborated these ndings of reduction in the bio lm formation in presence of dextransucrase antibody. These observations are in line with the results of antibio lm formation of secretary immunoglobulin A as reported by Huang et al. 30 and rPAc-speci c IgG reduced signi cantly bio lm formation in S. mutans challenged rats described by Sun et al. 31 Li et al. 32 have shown that adherence to tooth surface, bio im formation and growth of S. mutans involves a range of genetic networks which have a signi cant role to interact and coordinate to various environmental signals like surface oxygen, cell density carbon source pH and nutrient availability 33 .
The genes associated with bio lm formation have been reported by Bhagwat et al. 34 . In this study quantitative real time PCR analysis was used to quantify and compare the expression of GtfB, GtfC, BrpA, relA, Smu630, vicK genes. These genes help in bacterial adherence and bio lm formation 35,36,37 . The expression of various genes tested was down regulated when S. mutans were grown in presence of dextransucrase antibody. There was a 0.5 fold and 0.13 fold reduction in the expression of GtfB and GtfC genes respectively which have an important role in glucan formation from carbohydrates 38 . The observed reduction in the expression of these genes may result in suppression of bio lm formation, cell adhesion and integrity of cell wall. Similarly BrpA which is involved with the oxidative stress, acid tolerance and bio im formation has shown 0.16 fold reduction in its expression 39  Adherence of bacteria to the saliva coated surfaces is essential for the genesis of dental caries 42 .
Inhibition of adherence factors could be an effective measure to prevent progression of dental caries 43 .
The present ndings showed that dextransucrase antibody reduced the adherence of S. mutans by 58% as compared to control when grown in presence of 28µg of antibody for 20 hours. These results are similar to the ndings of Huang et al. 31  tooth surface as a result of its demineralisation 50 , leading to the progression of dental caries 51 . In the present study, there was essentially no effect of dextransucrase antibody on the acid production by S. mutans when the pH of the culture was measured at 0h and at 24h in the absence and presence of dextransucrase antibody. These experiments were carried out in presence of 28µg of antibody containing 5mL of media. It is likely that antibody concentration may be small in a large volume of the media than that used in other experiments. Thus no effect on acid production by S. mutans could be observed under the experimental conditions. Also it is likely that under acidic conditions, the enzyme antibody become inactive to exhibit any detectable effect on pH of the culture medium. Thus high concentration of antibody may be needed to observe any change in acid production by S. mutans.
Various immunological interventions have been tried for the prevention of caries formation, using various surface molecules of S. mutans including synthetic, conjugate and DNA vaccines. Although showing promising anticaries effects, but their use was restricted of having cross reactivity with human heart tissues and skeleton muscles 52 . However western blot analysis revealed that antibodies against dextransucrase did not show any cross reactivity with human heart, liver, gall bladder and other mammalian tissues tested as described previously 16 . In the present study various human samples were tested for cross reactivity with dextransucrase antibody by immunohistochemistry which also showed negative results con rming no cross reactivity of dextransucrase antibodies with the human tissues.
In conclusion the present study demonstrates that dextransucrase antibodies show an inhibitory effect on bio im formation, hydrophobicity, exopolysaccharide synthesis and cell adherence and has no cross reactivity with human samples. Thus the present data further supports the contention that dextransucrase is a potential candidate as a useful anticariogenic agent. Further studies on developing the monoclonal antibodies or sub unit fragments using dextransucrase protein may be of interest to explore the anticariogenic potential for clinical or physiological aspects.  Figure 1 Immuno-gold micrograph of S. mutans cells. Electron-dense immunogold particles (black arrow heads) are associated with the bacterial surface and a thin distribution of immuno-gold particles are present in the cytosolic region. Images were taken at magni cation X29000 bar, 100 nm. The sections were examined using a Tecnai, G 20 (FEI) transmission electron microscope operated at an accelerating voltage of 200 kV.  Fluorescence images of bio im formation by S. mutans in presence and absence of dextransucrase antibody. The images were taken at 10X, 20X and 40X using Evos FL Auto uorescent microscope and scale bar represents 400μm, 200μm, 100μm in length. The experiment was carried in triplicates.