【作者】 李华军；

【导师】 马郁芳；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 龋病（Dental caries）是发生在牙齿硬组织（牙釉质）的细菌感染性疾病。该病在全球发病率高、流行范围广，是严重危害人类健康的主要口腔疾病之一。长期以来，经过各个国家的努力，采取各种方法预防龋病的发生，特别是氟化物的应用，使龋病的预防收到了明显的效果。但是目前的控制措施还未能完全有效地控制龋病的发生。同时由于氟化物的长期使用，出现了耐氟菌株，这使龋病的预防形势更加严峻。这就迫切需要人们开发新的预防龋病的措施及治疗药物。链球菌群中的变形链球菌（Streptococcus mutans）为主要致龋菌。变形链球菌引起龋病的先决条件是其必须在牙齿表面定植，形成生物膜（Biofilm）即牙菌斑（Dental plaque）。定植在牙菌斑内的变形链球菌可以发酵蔗糖产酸，使牙釉质脱矿。形成生物膜被认为是多种口腔致病菌致病因素之一。因此寻找可以抑制变形链球菌及其生物膜的方法，是龋病预防、治疗的有效措施。抗菌肽（Antimicrobial peptides，AMPs）是具有抗菌（包括细菌、真菌）活性短肽的总称，具有广泛的杀菌活性。细菌细胞壁是维持细菌生存所必需的基本结构之一，与真核细胞不同，其主要的化学成分是肽聚糖。肽聚糖合成中的一个关键反应是UDP-N-乙酰葡糖胺（UDP-GlcNAc）与磷酸烯醇式丙酮酸（PEP）转化为UDP-N-乙酰基-3-O-（1-羧基乙烯基）-D-葡糖胺（UDP-N-乙酰葡糖胺烯醇丙酮酸，UDP-Glc-NAc-EP）。MurA（UDP-N-乙酰葡萄糖胺烯醇式丙酮酸转移酶）是催化此反应的酶，因此它是肽聚糖合成过程中的一个关键酶。由于此反应途径无代谢旁路，并且在人体中不存在此种代谢途径。MurA酶可能是研制预防龋病药物的作用靶点之一。因此，本课题的研究工作包括两部分。（1）筛选针对致龋性变形链球菌及其生物膜有效的抗菌肽，并揭示其作用机制。（2）针对变形链球菌细胞壁肽聚糖合成关键酶MurA作为药物靶点，通过克隆表达得到MurA蛋白，对其进行酶促动力学研究，建立高通量筛选MurA酶抑制剂的分子模型。第一部分取得的研究结果：1.抗菌肽对S. mutans的最低抑菌浓度（MIC）应用96孔板微孔培养基稀释方法测定三种抗菌肽（P-113、PAC-525、D-Nal-Pac-525）对S. mutans的MIC。MIC分别为P-113＞16μg/ml，PAC-5258μg/ml，D-NAL-PAC-5254μg/ml。确定最佳抗菌肽为D-Nal-Pac-525。2. D-Nal-Pac-525对S. mutans的生长抑制作用将不同浓度的D-Nal-Pac-525加入到对数期初期的S. mutans培养液中，于不同时间点取样，测OD600，绘制生长曲线。结果显示，当D-Nal-Pac-525浓度为2μg/ml S. mutans生长趋势没有变化，但当其浓度升高到4μg/ml S. mutans生长受到明显抑制。3. D-Nal-Pac-525对S. mutans的杀菌活性将D-Nal-Pac-525（终浓度分别为1、2、4μg/ml）加入到S. mutans培养液中（¹×108CFU/ml），厌氧培养，分别在2、4h取样，进行活菌计数，当D-Nal-Pac-525浓度在4g/ml时，与未加药的对照相比S. mutans活菌数量显著减少。4.扫描电镜观察D-Nal-Pac-525引起S. mutans形态学的变化将D-Nal-Pac-525加入到S. mutans（¹08CFU/ml）培养液中，至终浓度为4μg/ml。37oC厌氧培养4h。离心后收集菌体，标本经过处理固定后用SEM进行观察。与未处理的对照组相比，D-Nal-Pac-525处理后的S. mutans呈现明显的形态学变化。处理组细菌菌体明显变长，菌体表面粗糙有皱褶。同时在处理组的照片上观察到细菌崩解之后形成的碎片。5.透射电镜观察D-Nal-Pac-525引起S. mutans结构的变化同样将D-Nal-Pac-525加入到S. mutans（¹08CFU/ml）培养液中，至终浓度为4μg/ml。37oC厌氧培养4h。离心后收集菌体，标本经过处理固定后用TEM进行观察。D-Nal-Pac-525对S. mutans菌体表面结构的破坏作用。镜下未处理组菌体表面结构均一，呈高密度线。处理组菌体表面结构变模糊，甚至遭到破坏。同时在处理组菌体内部出现了高密度区域、细菌染色体凝集现象，胞内物质凝集及菌体细胞质膜破坏的现象。6. D-Nal-Pac-525抑制S. mutans生物膜的形成在PVC96孔板上建立S. mutans生物膜模型。将不同浓度的D-Nal-Pac-525（终浓度为0.25，0.5，1，2，4μg/ml）与S. mutans（¹×105CFU/ml）BHI培养基菌液（含3%蔗糖）共培养。结果显示D-Nal-Pac-525在浓度为2g/ml时可以抑制S. mutans生物膜的形成。OD600结果验证了肉眼观察的结果。D-Nal-Pac-525对已经形成的生物膜没有破坏作用。7.对S. mutans生物膜相关基因的转录水平没有影响经过D-Nal-Pac-525（4μg/ml）处理4h，S. mutans生物膜相关基因（brpA、vicR及gbpA）的转录水平没有发生变化。第二部分取得的研究结果：1.表达载体pET16b-Smu murA的构建从S. mutans UA159菌株基因组数据库中查询出变形链球菌（UA159）murA基因（SMU₁525）的核苷酸序列（大小为1272bp）。设计PCR引物，在上、下游引物的5’端分别加入Nde I和Xho I限制性内切酶位点。以S. mutans UA159基因组DNA为模板，扩增出S. mutans murA基因。将PCR产物与pMD18T克隆载体连接，再将其转化入感受态大肠杆菌Novablue中。用限制性内切酶Hind Ⅲ和EcoR I酶切的方法鉴定重组质粒。对pMD18-Smu murA中的murA基因进行DNA序列测定。将所测得的核苷酸序列与S. mutans UA159murA （SMU₁525）基因进行序列比对，完全一致。说明在本实验中获得的murA为正确的S. mutans UA159murA基因。再用Nde I、Xho I双酶切pMD18-Smu murA质粒。回收、纯化murA基因，连接到pET16b表达质粒的Nde I和Xho I位点，构建pET16b-Smu murA表达载体。用EcoR I酶鉴定阳性重组质粒。2. S. mutans MurA蛋白在大肠杆菌BL21（DE3）中的表达、纯化将pET16b-Smu murA表达载体转入大肠杆菌BL21（DE3）中。在37oC振荡培养3小时，达到对数生长期。然后加入终浓度为0.5mM IPTG，室温诱导细菌8小时，诱导携带pET16b-Smu murA表达载体的BL21（DE3）菌株表达重组MurA蛋白。MurA蛋白的N端与质粒pET16b上的组氨酸标签形成融合蛋白。用超声破碎诱导后的BL21（DE3）。分别对上清、沉淀组分进行SDS-PAGE和Westernblotting，结果表明S. mutans MurA蛋白在BL21（DE3）菌株中可溶性表达。用组氨酸-Ni2+亲和层析技术纯化MurA蛋白。对蛋白进行蛋白定量（考马斯亮蓝法），其中第2管MurA蛋白的浓度为1150μg/ml。SDS-PAGE和Westernblotting结果表明MurA蛋白的纯度较高。3. S. mutans MurA酶活性测定方法的建立（1）高效液相色谱法（HPLC）：用Nova-Pak C18色谱柱，以三乙胺-醋酸缓冲液为流动相，在260nm处检测反应底物UDP-GlcNAc的减少。（2）化学显色法：S. mutans MurA催化反应的产物之一为磷酸，磷酸可以与钼酸铵形成磷钼酸复合物后使孔雀石绿颜色由黄绿变为蓝绿。用酶标仪在620nm处检测吸光值变化，以测定所生成磷酸的含量。4. S. mutans MurA蛋白酶促反应动力学特性的研究反应底物UDP-GlcNAc和PEP与不同浓度MurA在37oC反应不同时间。结果表明S. mutans MurA反应初速度酶浓度范围为1.84μg/ml，时间范围为5min。分别改变反应的温度和pH值，利用酶标仪在620nm处吸光度值的变化，计算反应产物的生成量。确定S. mutans MurA酶蛋白的最适反应温度是37oC，最适pH值是7.5。采用最佳反应条件，保证一种底物过量，改变另一种底物浓度采用双倒数法测其Km值和Vmax。37oC，pH7.5，酶浓度为1.84μg/ml，反应时间为5分钟。分别用不同的底物浓度，进行酶促反应。利用酶标仪检测620nm处吸光度值，反应产物的生成量。用双倒数作图法得出S. mutans MurA的Km值和Vmax。对于底物PEP，Km值为0.086±0.001mM，Vmax为0.098±0.001mM min-1mg-1。对于底物UDP-GlcNAc的Km值为0.120±0.005mM，最大速率Vmax为0.048±0.002mM min-1mg-1。5.磷霉素对S. mutans MurA功能的验证用磷霉素（Fosfomycin）进一步鉴定纯化的S. mutans MurA功能。将一定浓度的磷霉素与MurA蛋白在室温下预孵（preincubation）10min后，检测MurA蛋白活性变化。MurA酶的活性受到磷霉素抑制。在UDP-GlcNAc存在情况下，MurA受到的抑制作用更加明显。从而证明S. mutans MurA具有UDP-N-乙酰葡萄糖胺烯醇式丙酮酸转移酶活性。结论：1.筛选的抗菌肽D-Nal-Pac-525可以抑制变形链球菌的生长及生物膜的形成，D-Nal-Pac-525可能成为新的预防龋病药物。2.构建了高表达S. mutans MurA蛋白的工程菌株，可以获得大量可溶性MurA蛋白。3.建立了快速、准确测定MurA酶活性的方法，并建立了高通量筛选MurA酶抑制剂的分子模型，为小分子抑制剂的筛选提供了物质保障。更多还原

【Abstract】 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.更多还原