Role Of SMU.2055 Gene In Regulating Acid Resistance Of Streptococcus Mutans UA159

Background and objectiveStreptococcus mutans,the primary etiological agent of human dental caries,exists almost exclusively in biofilms on tooth surfaces.The pathogenic potential of S.mutans is attributable to potent biofilm-forming abilities,a high capacity to produce acids,a high degree of acid tolerance.Streptococcus mutans is the most aciduric and also the most cariogenic,play a critical role in the onset and progression of dental caries.Acid resistance is the key survival mechanism for S.mutans to adapt to the dynamic acid environment of dental plaque,but its molecular mechanism is not yet fully understood.With the development of various modern molecular biology technology,more and more genes related to the acid resistance of S.mutans and their related proteins have been found,which lay the foundation for further elucidate the mechanism of acid resistanceProtein post-translational modifications play an important role in life activities of organisms,and most proteins undergo post-translational modifications.Protein acetylation was one of important modifications,and involved in almost every aspects of biological activities,stress response,central metabolism and protein synthesis and degradation,etc.lysine acetylation regulated colony morphology,biofilm formation,lipid synthesis,heat shock response,immunogenicity and carbon metabolism.The SMU.2055 gene from the S.mutans genome encodes a putative acetyltransferase protein with 163 amino-acid residues.The functionally characterized this kind of enzyme have been shown to catalyze the transfer of an acetyl group from acetyl-coenzyme A(Ac-CoA)to the amino group on the substrate and selectively acetyl one of the 4-5 amino groups.Some scholars have found that acetylation of lysine can stimulate Escherichia coli to resist different environmental stimuli,and the increase of acetyltransferase will not only increase the cell concentration,but also improve the stress and heat resistance ability of E.coli.The study shows that the regulation of protein acetylation can affect the structure and permeability of cell wall,and influence the formation of bacterial morphology and biofilm formation.In the early stage,we have analyzed the three-dimensional crystal structure of SMU.2055 protein through gene cloning,protein purification and crystal screening.The structure is included in PDB(protein data bank),and is numbered 3LD2.Moreover,five small molecule compounds,whose structure matched with that of the target protein in high degree,were designed and selected.These five compounds considered as effective inhibitors to SMU.2055.The virtual model of small molecule inhibitors we built will lay a foundation to the anticaries research based on the crystal structure of proteins.However,there is still a lack of research on the function of SMU.2055 gene at home and abroad.The purpose of this study is to observe the changes in acid tolerance of SMU.2055 deficient strains and the factors that may affect their acid tolerance,and to explore the role of SMU.2055 gene in acid resistance of S.mutans,to explore the function of SMU.2055 gene,is of great significance to the effective target for prevention of dental caries.Chapter 1 Construction of SMU.2055 diffcient mutant strainObjective:To construct SMU.2055 deletion mutant strain.Methods:Based on the whole genome sequence of wild S.mutans UA159,provided by NCBI,using Primer Premier 5.0 software design and synthesis the upstream and downstream primers of SMU.2055 gene.The upstream and downstream fragment were amplified by PCR,using restriction endonuclease BamH I and Hind Ⅲ,following proper restriction enzyme digestions,the flanking regions were cloned into two multiple cloning sites of plasmid pFW5 to generate recombinant plasmid.Subsequently,the recombinant plasmid was used to transform the wild-type strain UA159,which resulted in replacement of the SMU.2055gene by a non-polar spectinomycin resistance(Spe)marker via allelic exchange.The transformation was carried out in BHI medium in the presence of competence-stimulating peptide(CSP).The positive clones were screened by selective BHI medium,and the genomic DNA of the positive clones was extracted by PCR and sequencing.Results:After PCR identification and sequence confirmation,SMU.2055-deletion mutant were constructed successfully.Chaper 2 Effect of SMU.2055 gene on the acid resistance of S.mutansObjective:Scanning electron microscopy and transmission electron microscopy were used to observe the morphological structure of wild-type S.mutans UA159 and SMU.2055 defective strains.In order to explore ability of growth in both normal and acidic conditions,comparison of the killing pH,the glycolytic pH drop,the proton permeability,the cell permeability H+-ATPase activity,and the ability of biofilm formation between the SMU.2055 mutant and the wild S.mutans UA159.To explore the role of SMU.2055 gene in the acid resistance of S.mutans,and to further study the function of SMU.2055 gene.Methods:1.Scanning electron microscope observation.After the S.mutans UA159 and SMU.2055 gene defective strains were resuscitation,the bacterial solution was adjusted to OD600≈0.8,1:100 was diluted,37℃ anaerobic culture 16 h,the bacteria solution was abandoned,the cover glass was removed,2%glutaraldehyde was fixed 1 h,the ethanol gradient dehydration 30 min,drying and spraying gold,scanning electron microscope observation bacteria under scanning electron microscope.2.Electron microscope observation.After the S.mutans UA159 and SMU.2055 gene defective strains were resuscitation,the bacterial liquid OD600≈0.8 was adjusted,and the liquid medium was added to the liquid medium at 1:100,37℃ anaerobic to logarithmic growth period,3,000 r/min centrifugation,0.3%glutaraldehyde preimmobilized 15~30 min,10,000 r/min centrifugation 15 min,2%glutaraldehyde fixed,1%four oxygen.Osmium re immobilized,acetone gradient dehydrated,embedded,sectioning,uranium acetate and lead citrate double staining,transmission electron microscope observation.3.To explore the growth of two strainsin in different pH conditions.Overnight cultures of S.mutans UA159 and SMU.2055 mutans strains were inoculated into fresh BHI culture medium(pH7.5 or pH 5.5)and growth kinetics were monitored at 37℃for 24 h under anaerobic conditions respectively at 1 h intervals.4.The killing pH assay.Overnight cultures of S.mutans UA159 and SMU.2055 mutans strains were added to BHI liquid medium with pH value of 2.5-7.0 at 0.5 intervals respectively,incubated at 37℃ in an anaerobic atmosphere for 3 h and then diluted,plated on BHI agar,and incubated in an anaerobic atmosphere at 37℃ for 48h.5.Minimum glycolytic pH determination.Overnight cultures of S.mutans UA159 and SMU.2055 mutans strains were grown in BHI broth until mid-exponential phase(OD600 = 0.5).Cells were harvested,washed,and resuspended in 50 mM KC1,1 mM MgCl2,the pH was adjusted to 7.2 using KOH.Glucose Glucose was added to a final concentration of 1%(wt/vol)and the pH value was measured every 2 min for 30 min.6.Cell permeabilization assay.Samples of 25 mL of cultures were centrifuged in the cold,and cells from each sample were resuspended in 2.5 mL of 75 mM Tris-HCl buffer(pH 7.0)with 10 mM MgSO4.Toluene(250 μL)was added to each cell suspension prior to vigorous vortex mixing and incubation for 5 min at 37℃.Each cell suspension was then subjected to two cycles of freezing in a dry ice-ethanol bath and thawing at 37℃.Permeabilized cells were harvested by centrifugation.The protein concentration was measured using the Quick Start Bradford method.7.Proton permeability assay.Overnight cultures of S.mutans UA159 and SMU.2055 mutans strains were centrifugation,washed,resuspended in PBS buffer(pH 7.2),and incubated for 2 h.After incubation,cells were harvested and resuspended in 50 mM KC1 and 1 mM MgC12 to 20 mg/mL.Adjust pH to less than 4.7,and the pH was recorded.After 50 min,butanol was added to a final concentration of 10%(v/v),at 80 min,end-point pH was recorded.8.H+-ATPase activity assay.Cultures of S.mutans UA159 and SMU.2055 mutans strains were grown in BHI medium overnight.The prepared permeable cells(pH value of 7.5 or 5.5)were suspended in the 75 mM Tirs-HC1 buffer(pH 7.0,containing 10 mM MgSO4).The 3 mL bacteria liquid was taken and the same buffer solution was used as the blank control,and the bacterial liquid was measured by OD600.According to the relation curve of the cell dry weight and the absorbance,the concentration of the bacteria was calculated.The H+/K+-ATP enzyme assay kit was used for enzymatic reaction,and the activity of H+-ATPase was calculated.9.Biofilm formation analysis under different pH conditions.S.mutans UA159 and SMU.2055 mutans strains were incubated in BHI medium overnight.New fresh BHI medium((pH 7.5 or pH 5.5)cultures were inoculated by diluting them 1:100 and dispensing 5 mL aliquots into 6-well plates with coverslips in each well.After 24 h of 37℃ anaerobic incubation,the formed biofilms were washed gently twice with sterile PBS to remove unbound bacteria and stained with SYT09 for 15 min at room temperature in a dark room.Confocal laser scanning fluorescence microscope was used to examine.For the detection of S YT09(green),we used the 488 nm line of the argon laser.At least five independent fields were collected.10.The experiment was performed in triplicate.The results was analyzed by using SPSS 20.0 software.The statistical significance was defined as P<0.05.t-test was used to analyze the result.Results:1.Randomly selected a field of vision under SEM to observe its surface morphology,SMU.2055 gene defective plant morphology and obvious changes in substance between bacteria.The wild type S.mutans UA159 bacteria had regular morphology,smooth and complete envelope,spherical rod shape,and irregular substance between the bacteria.However,the SMU.2055 deficient bacteria were larger than those of the wild type,and some bacteria were distributed in lumps.2.TEM observation,wild type S.mutans UA159 had normal morphology,complete cell membrane structure,and two degree symmetry of bacteria under split state.SMU.2055 gene defective cell membrane is blurred,integrity is destroyed.3.The early growth capacity of the SMU.2055 deficient mutants decreased compared with that of the wild-type S.mutans UA159 in the BHI medium.While the defective strains had longer lag phase and slower growth speed.At pH 5.5,the OD600 of the two strains was significantly lower than that of pH 7.5.The growth of the strain was inhibited,and the growth of the defective strains was inhibited more obviously.After 20 h,there was no significant difference in the final concentration of the two strains at pH 7.5 and pH 5.5.4.With the decrease of the pH value,the viability of the two strains weakened gradually.The lethal pH value of wild type S.mutans UA159 was 3.5,and the lethal pH value of the SMU.2055 deficient mutants strain was 4.0.5.After adding glucose,S.mutans UA159 and SMU.2055 deficient strains could quickly metabolize substrate to produce acid,the pH value of the reaction system decreased rapidly.Before the 18 min,the pH value of UA159 was lower than that of deficient strain,early glycolytic rate was significantly higher than that of SMU.2055 deficient strain,statistically significant difference(P<0.05).However,SMU.2055 mutant strain did not show a detectable defect in the glycolytic machinery,as its minimum glycolytic pH did not differ from that of the parent strain,UA159.6.The protein concentration of wild type S.mutans UA159 was 1.2768 +0.2639 mg/mL,and the protein concentration of the SMU.2055 deficient strain was 1.6297 + 0.1249 mg/mL.Compared with the wild type,the cell permeability of the SMU.2055 deficient strain increased,and the difference was significant(P<0.001).7.Compared with the parental strain,the membrane of cells of strain SMU.2055 was more permeable to protons than the parental strain,as indicated by the slopes of the curves between 50 and 80 min.Following addition of butanol to cell suspensions,SMU.2055 deficient strain,exhibited a higher ApH to the parental strain.And SMU.2055 deficient strain also revealed an reduced terminal pH relative to strain UA159.8.At pH 7.5,compared with wild type S.mutans UA159,H+-ATPase activity of SMU.2055 deficient strain decreased slightly,and the difference was not statistically significant(P>0.05).At pH 5.5,the H+-ATPase activity of the SMU.2055 defective strain was significantly lower than that of the wild strain,and the difference was statistically significant(P<0.01).9.At pH 7.5 and pH 5.5,compared with wild-type S.mutans UA159,biofilm formation in SMU.2055 deficient strains were smaller,looser,and more pore structure.The ability of SMU.2055 defective strains to form biofilm was significantly lower than that of wild strain.At pH 5.5,the ability of two strains to form biofilms was higher than that of pH 7.5.The biofilms formed at that time were denser,and the bacterial masses were bigger,almost all over the whole field of vision.Conclusions:1.The SMU.2055 gene of S.mutans is associated with acid resistance.2.The deletion of SMU.2055 gene may affect the membrane structure of bacterial cells,affect the early growth,lethal pH value,cell permeability,proton permeability,H+-ATPase activity at pH 5.5 and biofilm formation ability of S.mutans under different pH conditions,but it has nothing to do with glycolysis ability.