Action Mechanism Of Antimicrobial Peptide On Streptococcus Mutans And Studies Of MurA As A Target

Oral disease is a major public health problem in high income countries. Theburden of oral disease is growing in many low-and middle income countries. Oraldisease is the fourth most expensive disease to treat.Dental caries is the disease of bacteia infections in the tooth enamel, it is themost common and costly oral disease caused by bacterial infection throughout theworld.For a long time, the efforts of the various countries had been taken to preventthe incidence of dental caries, in particular the application of fluoride cariesprevention received significant effect. But the current control measures has not beenfully effective in controlling the incidence of dental caries. In the same time due tothe long-term use of fluoride the fluoride resistant strains, which makes the sitution ismore severe dental caries prevention. There is an urgent need to develop newmeasures of prevention of dental caries and treatment drugs.Streptococcus mutans is the primary etiological agent of human dental caries. S.mutans can form biofilms （dental plaque） on the tooth surface, and then it usesmultiple fermentable sugars and then product acids. The acids cause dissolution ofminerals in tooth enamel and dental caries. Thus S. mutans and dental plaque arevaluable targets for dental caries prevention.Antimicrobial peptides （AMPs） are genetically common molecules of innateimmunity that have been discovered in single-cell and multicellular forms of life.AMPs are attractive future substitutes for conventional antibiotics as their killingactivity against a wide spectrum of microbiology （including bacteria and fugus）. Thebacterial cell wall which protects bacteria against osmotic pressure is responsible forthe cell shape. The bacterial cell wall is composed mainly of peptidoglycan. The cellwall of gram-positive bacteria is primarily composed of peptidoglycan （PG）. The biosynthesis of peptidoglycan is a complicated process. The first stage occurred in thecytoplasm is the formation of the N-acetylglucosamine-N-acetylmuramylpentapeptide, which is catalyzed by a series of Mur enzymes （MurA to MurF）. MurA,UDP-N-acetylglucosamine enolpyruvyl transferase, catalyzes the first step ofN-acetylglucosamine-N-acetylmuramyl pentapeptide biosynthesis.UDP-N-acetylglucosamine enolpyruvyl transferase （MurA） transfers the enolpyruvatefrom phosphoenolpyruvate （PEP） to the3-OH of UDP-N-acetylglucosamine（UDP-GlcNAc） to form UDP-Glc-NAc-EP. In some bacteria （e.g., E. coli）, MurA（UDP-N-acetylglucosamine enolpyruvyl transferase） catalyzes the first committedstep of peptidoglycan biosynthesis. MurA is an essential enzyme in E. coli, as itsdeletion is lethal to the organism, and it has no mammalian homolog. MurA of S.mutans will be a useful target to identified novel anti-cariogenic S. mutans agentstargeting peptidoglycan biosynthesis.The research works in the study inclued two parts.（1） To screen antimicrobialpeptides for cariogenic S. mutans and its biofilm and to reveal the action mechanismof an effective peptide.（2） To clone and express S. mutans MurA enzyme, which isinvolved in the biosynthesis of peptidoglycan, as a drug target; to study kinetics ofMurA enzyme and develop a molecular model of high-throughput screeninginhibitors of MurA.Results: The results of part1:1. The minimum inhibitory concentration （MIC） of D-Nal-Pac-525against S.mutans.The minimum inhibitory concentration （MIC） was determined by the standardbroth microdilution method. The assay was performed in a flat-bottom96wellmicrotiter plate （Nunc167008）. The MIC of D-Nal-Pac-525towards S. mutans wasexamined at concentrations of0.5-16g/ml. The MIC value of D-Nal-Pac-525was4g/ml.2. The inhibitory activity of D-Nal-Pac-525against S. mutans.The inhibitory activity of D-Nal-Pac-525against S. mutans was evaluated byOD610.The growth curve showed that D-Nal-Pac-525with robust inhibiting activityagainst S. mutans.3. The bactericidal activity of D-Nal-Pac-525against S. mutans.The bactericidal activity of D-Nal-Pac-525was performed using the brothdilution method by enumeration of viable organisms. The surviving cell numbers in D-Nal-Pac-525of4g/ml decreased dramatically compare to the control. Theseresults demonstrate that D-Nal-Pac-525has a dose-dependently and time-dependentlybactericidal effect.4. SEM of S. mutans treated by D-Nal-Pac-525SEM was used to observe morphological changes of the S. mutans treated withD-Nal-Pac-525. The S. mutans treated with D-Nal-Pac-525showed remarkablechanges in their cellular shape. The S. mutans treated with D-Nal-Pac-525lost thetypical shape and showed elongated spheres, which were significantly longer thannormal. S. mutans treated with D-Nal-Pac-525exhibited a rough cell surface withdiscrete ridges. The debris of cells also was observed in the images of S. mutanstreated with D-Nal-Pac-525.5. TEM of S. mutans treated by D-Nal-Pac-525TEM was used to observe the effect of D-Nal-Pac-525on S. mutansultrastructure. The S. mutans treated with D-Nal-Pac-525exhibited growth defects.Some of the treated cells’ membrane and wall became faint. Part of the treated cells’membrane and wall was even disrupted. The S. mutans treated with D-Nal-Pac-525showed cytoplasmic condensation, cytoplasmic membrane disruptions. TEM revealedthe S. mutans treated with D-Nal-Pac-525with nucleoid segregation, which has losttheir spatial organization in comparison to the untreated cells.6. Inhibitory activity of D-Nal-Pac-525against S. mutans biofilm formation.The biofilm formation was measured using a simple biofilm model in the wellsof a sterile96-well PVC （flexible） microplate （Costar2595）. D-Nal-Pac-525canprevent the formation of S. mutans biofilm at2g/ml concentration.7. Expression of biofilm related genes after treatment by D-Nal-Pac-525The results of RT-PCR indicated the transcription of biofilm related genes（brpA,vicR and gbpA） hadn’t change after treated by D-Nal-Pac-525.The results of part2:1. Construction of expression vector pET16b-Smu murADNA sequence of S. mutans murA（SMU₁525）gene （1272bp） was acquiredfrom S. mutans UA159genome database. One set of primers was designed. Nde I siteand Xho I site were added to5’ end of upstream primer and downstream primerrespectively to clone S. mutans murA into the Nde I site and Xho I site of pET16b. S.mutans murA gene was amplified from S. mutans genome by PCR.The purified PCR product was ligated into pMD18-T plasmid to construct pMD18-Smu murA. The constructed plasmid was transformed in NovaBluecompetent cells. Recombiant plasmid pMD18-Smu murA was confirmed by digestionof HindⅢ and EcoRI. S. mutans murA was sequenced and sequencing data wasanalyzed by Aliment. The results showed that S. mutans murA amplified by the PCRmethod was the correct gene. pMD18-Smu murA was digested by restrictionendonucleases Nde I and Xho I. S. mutans murA gene fragment was purified andligated into the Nde I and Xho I sites of pET16b. pET16b-Smu murA recombiantplasmid was confirmed by digestion of EcoRI.2. Expression of S. mutans MurA protein in E.coli BL21（DE3）pET16b-Smu murA plasmid was transformed into E.coli BL21（DE3） competentcells. The cells were cultured for3h at37℃. Then they were induced withIPTG atthe final concentration of0.5mM. Then the cells were cultured for8h at roomtemperature. BL21（DE3） cells after induction were sonicated by ultrasonic. TheN-terminus of MurA protein was fused with histidine tag in pET16b vector. Totalproteins from both supernatant and pellet fractions were analyzed by SDS-PAGE andWestern-blotting. The results showed that soluble S. mutans MurA protein wasproduced in BL21（DE3） cells.S. mutans MurA protein was purified by histidine-Ni2+affinity chromatography.The elution fraction2（1ml） was quantified （1150μg/ml） by Coomassie brilliant bluemethod.3. Establishment of enzyme assays for S. mutans MurA（1） HPLC: UDP-GlcNAc was separated with Nova-Pak C18（3.9×150mm,4μm） at a flow rate of0.5mLmin-1under20mM triethylamine-acetic acid buffer （pH4.0） and was monitored at260nm. The peak of UDP-GlcNAc was appearedapproximately at6.3min.（2） Colorimetric assays: inorganic phosphate is one production of S. mutansMurA enzymatic reation. The inorganic phosphate and ammonium molybdate reactand results in molybdenum blue. It turns malachite green from yellow to blue. Thevalue at620nm was detected.4. Determination of kinetic parameters of MurA proteinThe reaction was performed with UDP-GlcNAc, PEP and differentconcentration of S. mutans MurA. The results showed the initial velocity that theconcentration of MurA was1.84μg/ml and the incubation time was5minutes.The optimal temperature of S. mutans MurA is37oC. Te optimal pH of S. mutans MurA is7.5.By using the above assay in the initial velocity and optimal conditions, onesubstrate was at a saturated state and the concentration of the other one was changed.Km and Vmax of S. mutans MurA were calculated by the double-reciprocal plotmethod. The Km and Vmax for UDP-GlcNAc is0.120±0.005mM and0.048±0.002mM min-1mg-1respectively. For PEP Km and Vmax is0.086±0.001mM,0.098±0.001mM min-1mg-1, respectively.5. Confirmation of S. mutans MurA function by fosfomycinS. mutans MurA was inactivated by fosfomycin. The inhibition of MurA byfosfomycin is enhanced on preincubation with UDP-GlcNAc. The inhibition offosfomycin is a dose-dependent effect.Conclusions:1. Antimicrobial peptides D-Nal-Pac-525can inhibit the growth of S. mutansand its biofilm formation, so it may be a new candidate for dental caries prevention.2. Establish the MurA engineering strain, can produce abundant soluble MurAprotein.3. Erect the enzymatic assay for analysis of MurA protein function. Thehigh-throughput method will facilitate the screening of small molecule inhibitor toMurA.