| Background and objectiveDental caries is a chronic infectious disease,occurring worldwide,eventually causing tooth loss and pain.A variety of factors,especially the microbiome,interact to contribute to the onset and development of dental caries.Microflora can colonize tooth or mucosal surfaces,even forming biofilms,and their virulence plays an important role in acid formation,acidophilicity,and the production of extracellular polysaccharides(EPS).Therefore,the progress of dental caries is principally related to bacterial metabolism.Among the oral microbiome,Streptococcus mutans(S.mutans)is the primary pathogen in the etiology of dental caries in children and adults.Normally,S.mutans has the ability to survive and reproduce at acidic pH,and could regulate the activity of specific proteins associated with biofilm formation.Carbohydrate uptake and metabolism both play important roles in the virulence of S.mutans as it is particularly well adapted for the high-affinity,high-capacity catabolism of multiple carbohydrate sources.The predominant route for carbohydrate uptake by S.mutans is the phosphoenolpyruvate sugar phosphotransferase system(PTS).The PTS system minimally consists of Enzyme ?(E?)and HPr,which participate in a phosphotransfer reaction to a variety of Enzyme ?(E?)permeases that concomitantly phosphorylate and internalize diverse carbohydrate.S.mutans encodes as many as fifteen different E? permeases,composed of A,B,C,and occasionally D,domains.The Ells both translocate substrates into the cell,and act as stimulus sensors and chemoreceptors whose stimuli are transduced through HPr.E? and HPr are encoded by pts?(SMU.675)and ptsH,respectively.PtxA and PtxB,encoding the E? A and B domains,constitute the L-ascorbate specific PTS.The crystal structures of both PtxA and PtxB have been reported.In a previous study,ptxA and ptxB,which encode PtxA and PtxB,have been shown to be associated with the virulence of dental caries,especially with regard to acid production,acid resistance,EPS,and biofilm formation.PtxA/PtxB may therefore be an attractive target for inhibiting S.mutans and suppressing the translocation of L-ascorbate when at optimal oral ascorbate levels.Current research in dental caries has focused on identifying valid targets to inhibit the principal cariogenic microorganism,S.mutans.AMPs are widely applied in the treatment of dental caries,as alternative antimicrobial therapeutics,since they are able to selectively impact prokaryotes and only minimally trigger microbial resistance.Previous studies have shown that native AMPs isolated from bacteria,fungi,animals,and plants,can have significant bactericidal effects,however,they are expensive to produce and possess potential toxicity.As a result,modified AMPs have been developed with a lower production cost,a lower host toxicity,and a range of linear or cyclic forms,as well as other forms.It has also been reported that combinations of a traditional antibiotic and AMPs can be effective as antimicrobial agents against oral pathogens.Recently,the number of synthetic AMPs has increased in the field of oral diseases,especially those directed against S.mutans.In addition,specific or selectively targeted antimicrobial peptides(STAMPs)have been designed as structural AMPs that combine an S.mutans targeting domain with a killing domain.The mechanisms of AMPs are adapted for its permeabilization,or intracellular microbial functional proteins,DNA,RNA synthesis targeting,certain intracellular target interaction.Existing AMPs are basically targeting the membrane,however,it may widely inhibit bacteria,further breaking the imbalanced oral microbial ecology,causing a secondary infections.Thus,STAMP,towards S.mutans should still be searched necessarily.The goal of this study was to identify a novel membrane interacting AMP that targeted the PtxA/PtxB component of PTS and was capable of inhibiting S.mutans.A series of short lipopeptides was designed,wherein a fatty acid lipid tail was conjugated to the N-terminus of various tryptophan(W),lysine(K)and arginine(R)containing peptidyl heads,and these were evaluated for their antimicrobial activities.The preliminary mechanisms of action of these lipopeptides have been previously shown to be high affinity membrane binding or DNA binding.A novel AMP,targeting PtxA/PtxB,was identified by virtual screening using a structure-based docking analysis,and this was then used to investigate the effect on mutans and explore its mechanism of action.The antimicrobial activities against the wild-type(WT)S.mutans strain UA 159,a ptxA deletion mutant(?ptxA)/a ptxAB deletion mutant(?ptxAB),and a complemented ptxA deletion mutant strain(CptxA)/a complemented ptxAB deletion mutant strain(CptxAB)were compared to examine whether the AMP identified precisely targeted PtxA and PtxB.The mechanism was defined by comparing the expression of genes related to biofilm formation,and those in the L-ascorbate regulated operon as well as other PTS components,including gtfBC,which encodes enzymes that produce water-insoluble glucans;comDE and luxS,which encode components of the quorum-sensing(QS)system;sgaT,ptxB,ptxA,SMU.273,SMU.274,SMU.275,which are in the operon regulated by L-ascorbate,and SMU.675 and ptsH which encode other subunits of the PTS,and by determining the capability of PtxA and PtxB that transports extracellular L-ascorbate into cells.Chapter 1 The screening of novel membrane-targeted AMPs specially inhibiting PtxA/PtxB of Streptococcus mutansMaterial And Methods1.SYBYL X-2.0 was used for molecular docking as previously described.The 3D structures of PtxA and PtxB were obtained from the PDB database.Surflex-Docking results were evaluated using Total-Score,the threshold of which was set at 5 kcal/mol to capture the better docked molecules.2.The method used in this study using a standard 9-fluorenylmethoxy carbonyl(Fmoc;GL Biochem,Shanghai,China)solid phase protocol on a Rink Amide MHBA resin.Peptide purity was analyzed using reverse-phase high performance liquid chromatography(RP-HPLC).3.The minimum inhibitory concentration(MIC)were measured.The exponential phase cultures of S.mutans UA 159 and L.acidophilus ATCC 4356,E.coli ATCC 25922,S.aureus ATCC 12600 were diluted to 105 CFU/mL.MICs were determined as the lowest concentration of peptide that inhibited bacterial growth by visual inspection after 24 hours of incubation.4.The hemolytic activity was assessed using a modified protocol.995?L of PBS solution containing serial diluted peptides or 1%Triton X-100 was added to 5 ?L of rabbit erythrocytes,then incubated for 30 min at 37?,respectively.Following centrifugation,the supernatant was diluted five-fold with PBS and the OD415 was measured using a spectrophotometer.5.Cytotoxicity of C10-KKWW or C14-KKWW was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Oral periodontal ligament fibroblast cells(PDLCs)were grown in DMEM containing Cells were either treated with various concentrations of peptides or without peptides.Following 24 h,48 h and 72 h of incubation with peptides,10 ?L of 5 mg/mL MTT solution reagent was added to each well and incubated for 4 h.The purple-blue MTT formazan precipitate was dissolved in 150 mL of DMSO.The absorbance was determined using microplate reader at 490 nm.Experiments were run in triplicate.Results are expressed as a percentage of the inhibition rate for viable cells.6.The killing kinetics of the AMP against S.mutans were analyzed using a timekill assay as previously described.Final concentrations of 3.9,7.8,15.6,31.2,and 62.5 ?g/mL(0.5,1,2,4 and 8-fold higher than the MIC)AMPs were added to the bacterial suspension,with 1.9?g/mL(MIC)chlorhexidine(CHX)as the positive control,and a non-treated group as the negative control.At 0,5,30,60,120,and 180 min,the bacterial suspension was collected and diluted 102-,103-,or 104-fold and then immediately placed on ice.Following this 50 ?L of each dilution was spread on agar plates and the colonies were counted after 48 hours of anaerobic culture at 37?.All assays were repeated three times.7.MICs,hemolytic toxicity tests,and cytotoxicity tests were analyzed by one-way ANOVA analysis.P<0.05 represents a significant difference.Results1.C10-KKWW,C10-RIKWWK,C10-RWWR,C10-RWR,C10-KIKRWR,C10-KKWW,C14-RWR,C14-KKWW,C18-RWWR,C18-KWK were chosen as selectively AMPs targeted PtxA,and C10-KKWK?C10-RWR?C14-KKWW were chosen as selectively AMPs targeted PtxB with the total scores more than 5 kcal/mol.2.Peptide purity was analyzed using reverse-phase high performance liquid chromatography(RP-HPLC).The purities of them were more than 95%.3.The antibacterial activities of C10-RIKWWK,C10-KKWW,C14-KKWW were better whose MICs against S.mutans were 3.9 ?g/mL,7.8 ?g/mL,3.9 pg/mL,respectively.4.The hemolytic effects of C10-KKWW and C14-KKWW,over a range of concentrations,were significantly lower than those in the positive control(P<0.01).Even though the total-score of C10-RIKWWK was high,the toxicity of which was strong,even at a concentration of 1.9 ?g/mL.So we finally defined C10-KKWW and C14-KKWW to be further studied.5.Toxicity of C10-KKWW or C14-KKWW for human oral fibroblasts(HEPM)cells.The effective C10-KKWW and C14-KKWW concentrations only corresponded to a negligible cell viability reduction.6.The killing curve data revealed that the treatment with C10-KKWW at 15.6,31.2,and 62.5 ?g/mL for 5 min,however,C14-KKWW at 7.8,15.6,31.2 ?g/mL for 5 min,caused a decrease of approximately 1-1.5 log10 CFU/mL in S.mutans in a dose-dependent manner.Treated with 62.5 ?g/mL C10-KKWW for 1 h or 31.2?g/mL C14-KKWW for 30 min,105 CFU/mL of S.mutans were totally died.It's time-dose dependence.Chapter 2 The antimicrobial activities and target identification of C10-KKWW/C14-KKWWMaterials and Methods1.RT-PCR was used to examine the expression of the ptxA and ptxB gene after 3.75 mM,7.5 mM,15 mM,and 30 mM L-ascorbate was supplied to WT S.mutans(1×108 CFU/mL)for 1 hour,3 hours,and 5 hours,or after 7.4 mM sodium L-ascorbate was applied for 5 hours.The PCR products were analyzed by 2%agarose gel electrophoresis and Image J 2.0 was used to analyze the gray intensity of the PCR product bands.2.The MICs and MBCs for WT S.mutans,?ptxA/?ptxAB,and CptxA/CptxAB were measured using the method described above using three different culture conditions,namely 1)culturing in BHI,then supplied with 7.4 mM of sodium L-ascorbate for 5 hours and,finally treated with C10-KKWW/C14-KKWW;2)culturing in TVL,then treated with C10-KKWW/C14-KKWW;3)culturing in BHI,then treated with C10-KKWW/C14-KKWW.3.All bacterial strains were cultured in six-wells plate with coverslips in each well and formed biofilms.Biofilms were stained using the LIVE/DEAD BacLight Bacterial Viability Kit(L7012,Invitrogen,Carlsbad,CA,USA)and examined using an Olympus Fluoview FV10i confocal microscope(Olympus,Tokyo,Japan).Live cells were stained with SYTO 9(green fluorescence),and dead cells were stained with propidium iodide(PI)(red fluorescence).The areas stained green or red were quantitated using Image-Pro Plus 6.0.The live cell area/(live cell area+dead cell area)ratio was calculated as the%viability in each group.4.RT-PCR gray analysis and biofilm viability analysis were performed using one-way ANOVA.P<0.05 represents a significant difference.Results1.RT-PCR analysis was performed to confirm the optimal reaction time and concentration for L-ascorbate treatment in BHI medium that resulted in a higher expression of the ptxA/ptxB gene.the relative expression of the ptxA/ptxB gene when the cultures were supplemented with either 7.5 mM L-ascorbate,or 7.4 mM sodium L-ascorbate for five hours,was significantly higher than that in the other conditions tested(P<0.01).2.Compared to AptxA,in WT S.mutans and CptxA,C10-KKWW had a stronger MIC(3.9 ?g/mL).Compared to AptxAB,in WT S.mutans and CptxAB,C14-KKWW had a stronger MIC(1.9 pg/mL).3.The AptxA viability was significantly higher than the viability of WT S.mutans and CptxA,regardless of the culture condition used(P<0.05).The AptxAB viability was significantly higher than the viability of WT S.mutans and CptxAB,regardless of the culture condition used(P<0.05).4.C10-KKWW or C14-KKWW was more effective against WT S.mutans when L-ascorbate was added exogenously.Chapter 3 The possible mechanisms of C10-KKWW/C14-KKWW Materials and Methods1.Scanning electron microscopy was used to analyze bacterial morphology in the presence and absence of C10-KKWW/C14-KKWW,including S.mutans,L.acidophilus,E.coli and S.aureus.2.To identify the genes of sgaT,ptxB,ptxA,SMU.273,SMU.274,SMU.275,SMU.675 and pstH,regulated by C10-KKWW/C14-KKWW and confirm the underlying mechanism,qRT-PCR was performed.qRT-PCR was performed using a PCR machine(LC480,Roche,Basel,Switzerland)with a SYBR Premix ExTaq kit(Takara).S.mutans WT was cultured in TVL for 48 hours,treated with two different concentrations of C10-KKWW or C14-KKWW for 1 hour or 30 minutes,or cultured in BHI for sixteen hours,and then supplied with 7.4 mM sodium L-ascorbate for 5 hours and treated with C10-KKWW or C14-KKWW for 1 hour or 30 minutes.A non-treated group served as the negative control.3.To identify the genes of gtfB,gtfB,comD,comE and luxS,regulated by C10-KKWW/C14-KKWW and confirm the underlying mechanism,qRT-PCR was performed.S.mutans biofilms were cultured and formed in BHI for 24 hours,or in TVL for 120 hours,followed by treatment with C10-KKWW or C14-KKWW for 24 hours.A non-treated group served as the negative control.4.First,using HPLC,an ascorbate standard curve(using concentrations of 0.94 mM,1.87 mM,3.75 mM,7.50 mM,and 15.00 mM)or a sodium L-ascorbate standard curve(using concentrations of 0.90 mM,1.80 mM,3.70 mM,7.40 mM,14.80 mM)was generated.S.mutans WT was cultured in TVL for 48 hours,and then treated in the presence or absence of C10-KKWW for 1 hour or C14-KKWW for 30 min.WT S.mutans was also cultured in BHI supplymented with 7.4 mM sodium L-ascorbate for 5 hours and then treated with or without C10-KKWW for 1 hour or C14-KKWW for 30 min.The extracellular L-ascorbate or sodium L-ascorbate concentration before treatment was defined as the control.5.Recombinant PtxA was generated by a modified method.The DNA was synthesized and then inserted into the pET28a expression plasmid,and the correct ligation was verified by DNA sequencing.The final construct was transformed into E.coli BL21(DE3)for protein production.E.coli BL21(DE3)expressing PtxA/PtxB was grown in LB medium,and when the OD600 of the cell culture reached 0.6,protein expression was induced for 20 h by the addition of 0.5 mM IPTG(isopropyl ?-D-thiogalactoside).Expression of the His-tagged protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)of bacterial cell lysates.For purification,the clarified ultrasonic supernatant was applied to a nickel column and eluted sequentially.6.The SPR protocol has been described previously.Briefly,either interactions between PtxA and C10-KKWW or interations between PtxA/PtxB and C14-KKWW were analyzed using the PlexArrayr HT system.7.The relative expression of each gene in qRT-PCR and the concentration of ascorbic acid or sodium ascorbate in HPLC were analyzed by two independent samples t test,P<0.05,representing a significant difference.Results1.In the presence of C10-KKWW/C14-KKWW,there was some retention of normal membrane morphology,although the cell membranes were partly ruptured with amounts of debris being evident around the rough surfaces.2.L-ascorbate-induced operon genes,or other PTS genes,were significantly suppressed by C10-KKWW or C14-KKWW(P<0.05).3.The genes of GtfBC,comDE and luxS were significant elevated by the AMPs(P<0.05).4.The extracellular concentrations of L-ascorbate/sodium L-ascorbate were not changed before and after WT S.mutans treated with C10-KKWW or C14-KKWW.5.Either interaction between PtxA and C10-KKWW or interaction between PtxA/PtxB and C14-KKWW,the KD values of which both were lower than control groups.Conclusions1.C10-KKWW and C14-KKWW might be determined as the possible specially targeted AMP that inhibiting PtxA/PtxB.2.It was determined that L-ascorbate specific TVL medium(TV base medium with 15 mM L-ascorbate)or 7.4 mM sodium ascorbate for 5 h as a possible culture condition,activating L-ascorbate specific PTS,and activating function of PtxA,PtxB.3.The MICs and MBCs of C10-KKWW/C14-KKWW against AptxA/AptxAB were significantly higher than that of WT and CptxA/CptxAB;%viabilities of C10-KKWW/C14-KKWW against AptxA/AptxAB was significantly higher than that of WT and CptxA/CptxAB;The PtxA might be the target of C10-KKWW,PtxA and PtxB might be the targets of C14-KKWW.4.In conclusion,C10-KKWW or C14-KKWW has been developed that acts through interaction with the bacterial membrane and interferes with L-ascorbate translocation to inhibit S.mutans growth and eradicate its biofilm.5.When treated by C10-KKWW or C14-KKWW,S.mutans might compensate for the reduced GtfBC,comDE and luxS activity antagonized at the post-translational level.The decreased density of biofilm must be regulated by these genes to re-enhance density accumulation.Inhibited S.mutans might active the GTFs to synthesize EPS,promoting adhesion biofilm formation.6.C10-KKWW or C14-KKWW may be especially effective at optimal oral ascorbate levels.A combination of C10-KKWW or C14-KKWW with sodium L-ascorbate might also be a novel strategy for dental caries treatment. |
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