抗菌肽对变形链球菌作用机制以及MurA靶点的研究
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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(SMU_1525)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 (SMU_1525)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。
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-525was4
g/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-525
SEM 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-525
TEM 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-525
The 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 murA
DNA sequence of S. mutans murA(SMU_1525)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 protein
The 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 fosfomycin
S. 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.
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(SMU_1525)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 (SMU_1525)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。
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-525was4
g/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-525
SEM 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-525
TEM 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-525
The 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 murA
DNA sequence of S. mutans murA(SMU_1525)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 protein
The 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 fosfomycin
S. 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.
引文
[1] Petersen PE. The World Oral Health Report2003: continuous improvement oforal health in the21st century-the approach of the WHO Global Oral HealthProgramme. Community Dent Oral Epidemiol,2003,31(suppl.1):3-24
[2] Petersen PE. World Health Organization global policy for improvement of oralhealth--World Health Assembly2007. Int Dent J,2008,58(3):115-121
[3]周学东,岳松龄主编.实用龋病学.北京:人民卫生出版社,2008
[4]盛江药,刘正.变形链球菌耐氟菌株的诱导及产酸性的实验研究.中华口腔医学杂志,2000,35(2):95-98
[5] Marsh PD. Dental plaque: biological significance of a biofilm and communitylife-style. J Clin Periodontol,2005,32(Suppl.6):7–15
[6] Lemos JA, Abranches J, Burne RA. Responses of cariogenic streptococci toenvironmental stresses. Curr Issues Mol Biol,2005,7(1):95-107
[7] Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the naturalenvironment to infectious diseases. Nat Rev Microbiol,2004,2:95–108
[8] Carlsson J, Soderholm G, Almfeldt I. Prevalence of Streptococcus sanguis andStreptococcus mutans in the mouth of persons wearing full-dentures. Arch OralBiol,1969,14:243–249
[9] Chastre J. Evolving problems with resistant pathogens. Clin Microbiol Infect,2008,14(Suppl3):3-14
[10]Choi BK, Kim KY, Yoo YJ, Oh SJ, Choi JH, Kim CY. In vitro antimicrobialactivity of a chitooligosaccharide mixture against Actinobacillusactinomycetemcomitans and Streptococcus mutans. Int J Antimicrob Agents,2001,18(6):553-557
[11]Hancock RE, Patrzykat A. Clinical development of cationic antimicrobialpeptides: from natural to novel antibiotics. Curr Drug Targets Infect Disord,2002,2:79-83
[12]Riley MA, Wertz JE. Bacteriocins: evolution, ecology, and application. AnnuRev Microbiol,2002,56:117-137
[13]Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB,Primeaux C, Tian R, Kenton S, Jia H, Lin S, Qian Y, Li S, Zhu H, Najar F, Lai H,White J, Roe BA, Ferretti JJ. Genome sequence of Streptococcus mutansUA159--a cariogenic dental pathogen. Proc Natl Acad Sci U.S.A.2002,99(22):14434-14439
[14]Brown ED, Vivas EI, Walsh CT, Kolter R. MurA (MurZ), the enzyme thatcatalyzes the first committed step in peptidoglycan biosynthesis, is essential inEscherichia coli. J Bacteriol,1995,177:4194–4197
[15]da Silva BR, de Freitas VA, Nascimento-Neto LG, Carneiro VA, Arruda FV, deAguiar AS, Cavada BS, Teixeira EH. Antimicrobial peptide control ofpathogenic microorganisms of the oral cavity: a review of the literature.Peptides,2012,36(2):315-21
[1] Hancock RE, Patrzykat A. Clinical development of cationic antimicrobialpeptides: from natural to novel antibiotics. Curr Drug Targets Infect Disord,2002,2:79-83
[2] Riley MA, Wertz JE. Bacteriocins: evolution, ecology, and application.Annu Rev Microbiol,2002,56:117-137
[3] Merrifield EL, Mitchell S, Ubach J, Boman HG, Andreu D, Merrifield RB.D-enantiomers of15-residue cecropin A-melittin hybrids. Int J Pept ProteinRes,1995,46:214-220
[4] Oren Z, Ramesh J, Avrahami D, Suryaprakash N, Shai Y, Jelinek R. Modeof membrane interaction and structure in micelles of a short a-helical lyticpeptide and its diastereomer determined by NMR, FTIR, and fluorescencespectroscopy. Eur. J. Biochem,2002,269:3869-3880
[5] Wu JM, Wei SY, Chen HL, Weng KY, Cheng HT, Cheng JW. Solutionstructure of a novel D-β-naphthylalanine substituted peptide with potentialantibacterial and antifungal activities. Biopolymers,2007,88:738-745
[6] Clinical and Laboratory Standards institute. Performance standards forantimicrobial susceptibility testing: fifteenth informational supplementM100-S15. CLSI, Wanye PA, USA2005
[7] Liu N, Chen XG, Park HJ, Liu CG, Liu CS, Meng XH, Yu LJ. Effect ofMW and concentration of chitosan on antibacterial activity of Escherichiacoli. Carbohydrate Polymer,2006,64:60–65
[8] Liu H, Du Y, Wang X, Sun L. Chitosan kills bacteria through cellmembrane damage. Int J Food Microbiol,2004,95(2):147-155
[9] Stepanovic S, Cirkovic I, Ranin L, Svabic-Vlahovic M. Biofilm formationby Salmonella spp. and Listeria monocytogenes on plastic surface. LettAppl Microbiol,2004,38:428-432
[10]Russell RR. How has genomics altered our view of caries microbiology?Caries Res,2008,42:319-327
[11]Wilson M. Susceptibility of oral bacterial biofilms to antimicrobial agents. JMed Microbiol,1996,44:79-87
[12]Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA,Sahasrabudhe A, Dewhirst FE. Bacterial diversity in human subgingivalplaque. J Bacteriol,2001,183(12):3770-3783
[13]Marsh PD. Are dental diseases examples of ecological catastrophes?Microbiology,2003,149:279-294
[14]Cintas LM. Bacteriocins of Lactic Acid Bacteria. Food Scie Tech Int,2001,7(4):281-305
[15]Liu Y, Wang L, Zhou X, Hu S, Zhang S, Wu H. Effect of the antimicrobialdecapeptide KSL on the growth of oral pathogens and Streptococcusmutans biofilm. Int J Antimicrob Agents,2011,37(1):33-8.
[16]Wei GX, Campagna AN, Bobek LA. Effect of MUC7peptides on thegrowth of bacteria and on Streptococcus mutans biofilm. J AntimicrobChemother,2006,57:1100-1109
[17]Raj PA, Antonyraj KJ, Karunakaran T. Large-scale synthesis and functionalelements for the antimicrobial activity of defensins. Biochem J,2000,347(3):633-41
[18]Beckloff N, Laube D, Castro T, Furgang D, Park S, Perlin D, Clements D,Tang HZ, Scott RW, Tew GN, Diamond G. Activity of an antimicrobialpeptide mimetic against planktonic and biofilm culture of oral pathogens.Antimicrob Agents Chemother,2007,51(11):4125-32
[19]Zasloff M. Antimicrobial peptides of multicellular organisms. Nature,2002,415:389-395
[20]Cabrera JE, Caglier C, Quan S, Squires CL, Jin DJ. Active Transcription ofrRNA Operons Condenses the Nucleoid in Escherichia coli: Examining theEffect of Transcription on Nucleoid Structure in the Absence of Transertion.J. Bacteriol,2009,191:4180-4185
[21]Shemesh M, Tam A, Steinberg D. Differential gene expression profiling ofStreptococcus mutans cultured under biofilm and planktonic conditions.Microbiology,2007,153:1307-1317
[22]Chitnis SN and Prasad KS.Seminalplasmin, an antimicrobial protein frombovine seminal plasma, inhibits peptidoglycan synthesis in Escherichia coli.FEMS Microbiol Lett,1990,60:281-284.
[23]Wang Y, Lee SM, Dykes GA. Potential mechanisms for the effects of teaextracts on the attachment, biofilm formation and cell size of Streptococcusmutans. Biofouling,2013,29(3):307-18.
[1] Park JT, Neidhardt R, Curtiss III, Ingraham JL, ECC Lin, Low KB, Magasanik B. Escherichia coli and Salmonella: cellular and molecular biology,2nd ed.1996.vol.1. ASM Press, Washington, D.C.
[2] Barreteau H, Kovac A, Boniface A, Sova M, Gobec S, Blanot D. Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol Review,2008,(32):168-207
[3] Zhang WL, Jones VC, Scherman MS, Mahapatra S, Crick D, BhamidiS, Xin Y, McNeil MR, Ma YF. Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase. Int J Biochem Cell Biol,2008,40(11):2560-2571
[4] Clifton E, Barry, Dean C, Crick, Michael R. McNeil. Targeting the formation of the cell wall core of M. tuberculosis. Infectious Disorders Drug Targets,2007,7:1-21
[5] Brown ED, Vivas EI, Walsh CT, Kolter R. MurA (MurZ), the enzymethat catalyzes the first committed step in peptidoglycan biosynthesis, isessential in Escherichia coli. J Bacteriol,1995,177:4194–4197
[6] Marquardt JL, Siegele DA, Kolter R, Walsh CT. Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase. J Bacteriol,1992,174:5748-5752
[7] Wanke C, Falchetto R, Amrhein N. The UDP-N-acetylglucosamine1-carboxyvinyl-transferase of Enterobacter cloacae. Molecular cloning, sequencing of the gene and overexpression of the enzyme. FEBS Lett,1992,301:271-276
[8] Benson TE, Marquardt JL, Marquardt AC, Etzkorn FA, Walsh CT. Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase. Biochemistry,1993,32:2024-2030
[9] Benson TE, Harris MS, Choi GH, Cialdella JI, Herberg JT, Martin JP,Baldwin ET. A structural variation for MurB--X-ray crystal structure ofStaphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase(MurB).Biochemistry,2001,40:2340-2350
[10]钟启平.口腔微生物学,第一版,北京,人民卫生出版社,2009;165-173
[11]Kumar S, Parvathi A, Hernandez RL, Cadle KM, Varela MF. Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA)from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli. Arch Microbiol,2009,191:425-429
[12]Gunetileke KG, Anwar RA. Biosynthesis of uridine diphospho-N-acetylmuramic acid II. Purification and properties of pyruvate-uridine diphospho-N-acetylglucosamine transferase and characterization of uridine diphospho-N-acetylenopyruvylglucosamine. J Biol Chem,1968,243:5770-5778
[13]Mengin-Lecreulx D, Flouret B, van Heijenoort J. Pool levels of UDP N-acetylglucosamine and UDP N-acetylglucosamine-enolpyruvate in Escherichia coli and correlation with peptidoglycan synthesis. J Bacteriol,1983,154:1284-1290
[14]Eschenburg S, Priestman M, Schonbruns E. Evidence that the fosfomycin target Cys115in UDP-N-acetylglucosamine enolpyruvyl transferase(MurA) is essential for product release. J Biol Chem,2005,280:3757-3763
[15]DeSmet KA, Kempsell KE, Gallagher A, Duncan K, Young DB. Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis. Microbiology,1999,145:3177-3184
[16]McCoy AJ, Sandlin RC, Maurulli AT. In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involve in peptidoglycan synthesis and fosfomycin resistance. JBacteriol,2003,185:1218-1228
[17]Russell RR. How has genomics altered our view of caries microbiology?Caries Res,2008,42:319-327
[18]Lemos JA, Burne RA. A model of efficiency--stress tolerance by Streptococcus mutans. Microbiology,2008,154:3247-3255
[19]Blake KL, O'Neill AJ, Mengin-Lecreulx D, Henderson PJ, Bostock JM,Dunsmore CJ, Simmons KJ, Fishwick CW, Leeds JA, Chopra I. Thenature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors. Mol Microbiol,2009,72(2):335-43
[20]Han SG, Lee WK, Jin BS, Lee KI, Lee HH, Yu YG. Identification ofNovel Irreversible Inhibitors of UDP-N-Acetylglucosamine EnolpyruvylTransferase (MurA) from Haemophilus influenzae. J Microbiol Biotechnol,2013,23(3):329-34.
[1] Beutler B. Innate immunity--an overview. Molecular Immunology,2004,40:845–859
[2] Hancock RE and Sahl HG. Antimicrobial and host-defense peptides as newanti-infective therapeutic strategies. Nature Biotechnology,2006,24:1551–1557
[3] Gordon YJ, Romano ski EG, McDermott AM. A review of antimicrobialpeptides and their therapeutic potential as anti-infective drugs. Current EyeResearch,2005,30:505–515
[4] McPhee JB, Hancock RE. Function and therapeutic potential of host defensepeptides. Journal of Peptide Science,2005,11:677–687
[5] Yeaman MR, Yount NY. Unifying themes in host defense effecterpolypeptides. Nature Reviews Microbiology,2007,5:727–740
[6] Linde A, Ross CR, Davis EG, Dib L, Blecha F, Melgarejo T. Innate immunityand host defense peptides in veterinary medicine. Journal of VeterinaryInternal Medicine,2008,22:247–265
[7] Sang Y and Blecha F. Porcine host defense peptides--expanding repertoireand functions. Developmental and Comparative Immunology,2009,33(3):334-43
[8] Boman HG. Antibacterial peptides--basic facts and emerging concepts.Journal of Internal Medicine,2003,254:197–215
[9] Cotter PD, Hill C, Ross RP. Bacteriocins--developing innate immunity forfood. Nature Reviews Microbiology,2005,3:777–788
[10]Willey JM, Vander Dunk WA. Antibiotics--peptides of diverse structure andfunction. Annual Review of Microbiology,2007,61:477–501
[11]Hammami R, Zouhir A, Ben Hamida J, Fliss I. BACTIBASE--aweb-accessible database for bacteriocin characterization.BMC Microbiology,2007,7:89
[12]De Vuyst L, Leroy F. Bacteriocins from lactic acid bacteria--production,purification, and food applications.Journal of Molecular Microbiology andBiotechnology,2007,13:194–199
[13]Sit CS, Vederas JC. Approaches to the discovery of new antibacterial agentsbased on bacteriocins. Biochemistry and Cell Biology,2008,86:116–123
[14]Dufour A, Hindre T, Haras D, Le Pennec JP. The biology of antibiotics fromthe lactic in481group is coming of age. FEMS Microbiology Reviews,2007,31:134–167
[15]Crispie F, Twomey D, Flynn J, Hill C, Ross P, Meaney W. The antibioticlactic in3147produced in a milk based medium improves the efficacy of abismuth-based teat seal in cattle deliberately infected with Staphylococcusaurous.Journal of Dairy Research,2005,72:159–167
[16]Gardiner GE, Rea MC, O’Riordan B, O’Connor P, Morgan SM, Lawlor PG,Lynch PB, Cronin M, Ross RP, Hill C. Fate of the two-component antibioticlactic in3147in the gastrointestinal tract. Applied and EnvironmentalMicrobiology,2007,73:7103–7109
[17]Kruszewska D, Sahl HG, Bierbaum G, Pag U, Hynes SO, Ljungh A.Mersacidin eradicates methicillin-resistant Staphylococcus aureus (MRSA) ina mouse rhinitis model.Journal of Antimicrobial Chemotherapy,2004,54:648–653
[18]Duquesne S, Destoumieux Garzon D, Peduzzi J, Rebuffat S. Microclines,gene-encoded antibacterial peptides from enter bacteria. Natural ProductReports,2007,24:708–734
[19]Nes IF, Diep DB, Holo H. Bacteriocin diversity in Streptococcus andEnterococcus. Journal of Bacteriology,2007,189:1189–1198
[20]Rossi LM, Rangasamy P, Zhang J, Qiu XQ, Wu GY.Research advances in thedevelopment of peptide antibiotics. Journal of Pharmaceutical Sciences,2008,97:1060–1070
[21]Duclohier H. Peptaibiotics and peptaibols--an alternative to classicalantibiotics. Chemistry and Biodiversity,2007,4:1023–1026
[22]Leitgeb B, Szekeres A, Manczinger L, Vagvolgyi C, Kredics L. The history ofalamethicin--a review of the most extensively studied peptaibol. Chemistryand Biodiversity,2007,4:1027–1051
[23]Zhu S. Discovery of six families of fungal defending-like peptides providesinsights into origin and evolution of the C Salphabeta defenses. MolecularImmunology,2008,45:828–838
[24]Mygind PH, Fischer RL, Schnorr KM, Hansen MT, Sonksen CP, Ludvigsen S,Ravento′s D, Buskov S, Christensen B, De Maria L, Taboureau O, Yaver D,Elvig-J rgensen SG, S rensen MV, Christensen BE, Kjaerulff S,Frimodt-Moller N, Lehrer RI, Zasloff M, Kristensen HH. Plectasin is apeptide antibiotic with therapeutic potential from asaprophytic fungus. Nature,2005,437:975–980
[25]Silverstein KA, Graham MA, Paape TD, VandenBosch KA. Genomeorganization of more than300defensinlike genes in Arabidopsis. PlantPhysiology,2005,138:600–610
[26]Thevissen K, Kristensen HH, Thomma BP, Cammue BP, Francois IE.Therapeutic potential of antifungal plant and insect defensins. Drug DiscoveryToday,2007,12:966–971
[27]Zhang L, Dhillon P, Yan H, Farmer S, and Hancock RE. Interactions ofbacterial cationic peptide antibiotics with outer and cytoplasmic membranesof Pseudomonas aeruginosa. Antimicrob Agents Chemother,2000,44:3317–3321.
[28]Ireland DC, Wang CK, Wilson JA, Gustafson KR, Craik DJ. Cyclotides asnatural anti-HIV agents. Biopolymers,2008,90:51–60
[29]Barbeta BL, Marshall AT, Gillon AD, Craik DJ, Anderson MA. Plantcyclotides disrupt epithelial cells in the midgut of lepidopteran larvae.Proceedings of the National Academy of Science, USA,2008,105:1221–1225
[30]Colgrave ML, Kotze AC, Huang YH, O’Grady J, Simonsen SM, Craik DJ.Cyclotides--natural, circular plant peptides that possess significant activityagainst gastrointestinal nematode parasites of sheep. Biochemistry,2008,47:5581–5589
[31]Whittington CM, Papenfuss AT, Bansal P, Torres AM, Wong ES, Deakin JE,Graves T, Alsop A, Schatzkamer K, Kremitzki C, Ponting CP, Temple-SmithP, Warren WC, Kuchel PW, Belov K. Defensins and the convergent evolutionof platypus and reptile venom genes. Genome Research,2008,18:986–994
[32]Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting CP,Gru¨tzner F, Belov K. Genome analysis of the platypus reveals uniquesignatures of evolution. Nature,2008,453:175–183
[33]Ganz T. Defensins--antimicrobial peptides of innate immunity. NatureReviews Immunology,2003,3:710–720
[34]Selsted ME and Ouellette AJ. Mammalian defensins in the antimicrobialimmune response. Nature Immunology,2005,6:551–557
[35]Lehrer RI. Multispecific myeloid defensins. Current Opinion in Hematology,2007,14:16–21
[36]Lynn DJ, Bradley DG. Discovery of alpha-defensins in basal mammals.Developmental and Comparative Immunology,2007,31:963–967
[37]Brogden KA. Antimicrobial peptides--pore formers or metabolic inhibitors inbacteria? Nature Reviews Microbiology,2005,3:238–250
[38]Zaiou M. Multifunctional antimicrobial peptides--therapeutic targets inseveral human diseases. Journal of Molecular Medicine,2007,85:317–329
[39]Kang JH, Lee MS. Characterization of a bacteriocin produced byEnterococcus faecium GM-1isolated from an infant. Journal of AppliedMicrobiology,2005,98:1169–1176
[40]Svetoch EA, Eruslanov BV, Perelygin VV, Mitsevich EV, Mitsevich IP,Borzenkov VN, Levchuk VP, Svetoch OE, Kovalev YN, Stepanshin YG,Siragusa GR, Seal BS, Stern NJ. Diverse antimicrobial killing byEnterococcus faecium E50-52bacteriocin. Journal of Agricultural and FoodChemistry,2008,56:1942–1948
[41]Wiedemann I, Benz R, Sahl HG. Lipid II-mediated pore formation by thepeptide antibiotic Nisin--a black lipid membrane study. J Bacteriol,2004,186：3259–3261
[42]van Kraa CE, Breukink HS, Rollema R J, Siezen RJ, Demel RA, De Kruijff B,Kuipers OP．Influence of charge differences in the C-terminal part of Nisin onantimicrobial activity and signaling capacity. Eur J Biochem,1997,247：l14–120
[43]Huang HW. Molecular mechanism of antimicrobial peptides--the origin ofcooperativity. Biochimica et Biophysica Acta,2006,1758:1292–1302
[44]Jang H, Ma B, Woolf TB, Nussinov R. Interaction of protegrin-1with lipidbilayers--membrane thinning effect.Biophysical,2006,91:2848–2859
[45]Mani R, Cady SD, Tang M, Waring AJ, Lehrer RI, Hong M.Membrane-dependent oligomeric structure and pore formation of abeta-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR.Proceedings of the National Academy of Sciences, USA,2006,103:16242–16247
[46]Murakami M, Lopez-Garcia B, Braff M, Dorschner RA, Gallo RL.Postsecretory processing generates multiple cathelicidins for enhanced topicalantimicrobial defense. Journal of Immunology,2004,172:3070–3077
[47]Draper LA, Ross RP, Hill C, Cotter PD. Lantibiotic immunity, CurrentProtein and Peptide. Science,2008,9:39–49
[48]Scott MG, Dullaghan E, Mookherjee N, Glavas N, Waldbrook M, ThompsonA, Wang A, Lee K, Doria S, Hamill P, Yu JJ, Li Y, Donini O, Guarna MM,Finlay BB, North JR, Hancock RE. An anti-infective peptide that selectivelymodulates the innate immune response. Nature Biotechnology,2007,25:465–472
[49]Gunn JS. The Salmonella PmrAB regulon--lipopolysaccharide modifications,antimicrobial peptide resistance and more. Trends in Microbiology,2008,16:284–290
[50]Kraus D, Peschel A. Staphylococcus aureus evasion of innate antimicrobialdefense. Future Microbiology,2008,3:437–451
[51]Andres E, Dimarcq JL. Clinical development of antimicrobial peptidesInternational. Journal of Antimicrobial Agents,2005,25:448–449
[52]Bansal PS, Torres AM, Crossett B, Wong KK, Koh JM, Geraghty DP,Vandenberg JI, Kuchel PW. Substrate specificity of platypus venomL-to-D-peptide isomerase. Journal of Biological Chemistry,2008,283:8969–8975
[53]Jenssen H, Fjell CD, Cherkasov A, Hancock RE. QSAR modeling andcomputer-aided design of antimicrobial peptides. Journal of PeptideScience,2008,14:110–114
[54]Hyvonen P, Suojala L, Orro T, Haaranen J, Simola O, R ntved C, Pyorala S.Transgenic cows that produce recombinant human lactoferrin in milk are notprotected from experimental Escherichia coli intramammary infection.Infection and Immunity,2006,74:6206–6212
[55]Han ZS, Li QW, Zhang ZY, Yu YS, Xiao B, Wu SY, Jiang ZL, Zhao Hw,Zhao R, Li J. Adenoviral vector mediates high expression levels of humanlactoferrin in the milk of rabbits. Journal of Microbiology and Biotechnology,2008,18:153–159
[56]Zhang J, Li L, Cai Y, Xu X, Chen J, Wu Y, Yu H, Yu G, Liu S, Zhang A,Chen J, Cheng G. Expression of active recombinant human lactoferrin in themilk of transgenic goats. Protein Expression and Purification,2008,57:127–135
[57]Wu SC, Chen HL, Yen CC, Kuo MF, Yang TS, Wang SR, Weng CN, et al.Recombinant porcine lactoferrin expressed in the milk of transgenic miceenhances offspring growth performance. Journal of Agricultural and FoodChemistry,2007,55:4670–4677
[58]Chen HL, Wang LC, Chang CH, Yen CC, Cheng WT, Wu SC, Hung CM,Kuo MF, Chen CM. Recombinant porcine lactoferrin expressed in the milk oftransgenic mice protects neonatal mice from a lethal challenge withenterovirus type71. Vaccine,2008,26:891–898
[59]Humphrey BD, Huang N, Klasing KC. Rice expressing lactoferrin andlysozyme has antibiotic-like properties when fed to chicks. Journal ofNutrition,2002,132:1214–1218
[60]Salzman NH, Ghosh D, Huttner KM, Paterson Y, Bevins CL. Protectionagainst enteric salmonellosis in transgenic mice expressing a human intestinaldefensin.Nature,2003,422:522–526
[61]Lee PH, Ohtake T, Zaiou M, Murakami M, Rudisill JA, Lin KH, Gallo RL.Expression of an additional cathelicidin antimicrobial peptide protects againstbacterial skin infection.Proceedings of the National Academy of Sciences,USA2005,102:3750–3755
[62]Cheung QC, Turner PV, Song C, Wu D, Cai HY, MacInnes JI, Li J. Enhancedresistance to bacterial infection in protegrin-1transgenic mice. AntimicrobialAgents and Chemotherapy,2008,52:1812–1819
[63]Yamaguchi Y, Nagase T, Tomita T, Nakamura K, Fukuhara S, Amano T,Yamamoto H, Ide Y, Suzuki M, Teramoto S, Asano T, Kangawa K, NakagataN, Ouchi Y, Kurihara H. Beta-defensin overexpression induces progressivemuscle degeneration in mice. American Journal of Physiology–CellPhysiology,2007,292: C2141–C2149
[64]Peters BM, Shirtliff ME, Jabra-Rizk MA. Antimicrobial Peptides:PrimevalMolecules or Future Drugs? PLoS Pathog2010;6(10): e1001067.doi:10.1371/journal.ppat.1001067
[65]Guaní-Guerra E, Santos-Mendoza T, Lugo-Reyes SO, Teran LM.Antimicrobial peptides--general overview and clinical implications in humanhealth and disease. Clin Immunol,2010,135(1):1-11
[66] Chitnis SN and Prasad KS.Seminalplasmin, an antimicrobial protein frombovine seminal plasma, inhibits peptidoglycan synthesis in Escherichiacoli.FEMS Microbiol Lett,1990,60:281–284.
[67]Xiong YQ, Bayer AS, and Yeaman MR.Inhibition of intracellularmacromolecularsynthesis in Staphylococcus aureus by thrombin-inducedplatelet microbicidalproteins. J Infect Dis,2002,186:668–677.
[68]Park CB, Kim HS, and Kim SC. Mechanism of action of theantimicrobialpeptide buforin II: buforin II kills microorganisms by penetratingthe cell membraneand inhibiting cellular functions. Biochem Biophys ResCommun,1998,244:253–257.
[69]del Castillo FJ, del Castillo I, and Moreno F. Construction andcharacterization of mutations at codon751of the Escherichia coli gyrB genethat confer resistance to the antimicrobial peptide microcin B17and alter theactivity of DNA gyrase. J Bacteriol,2001,183:2137–2140.
[2] Petersen PE. World Health Organization global policy for improvement of oralhealth--World Health Assembly2007. Int Dent J,2008,58(3):115-121
[3]周学东,岳松龄主编.实用龋病学.北京:人民卫生出版社,2008
[4]盛江药,刘正.变形链球菌耐氟菌株的诱导及产酸性的实验研究.中华口腔医学杂志,2000,35(2):95-98
[5] Marsh PD. Dental plaque: biological significance of a biofilm and communitylife-style. J Clin Periodontol,2005,32(Suppl.6):7–15
[6] Lemos JA, Abranches J, Burne RA. Responses of cariogenic streptococci toenvironmental stresses. Curr Issues Mol Biol,2005,7(1):95-107
[7] Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the naturalenvironment to infectious diseases. Nat Rev Microbiol,2004,2:95–108
[8] Carlsson J, Soderholm G, Almfeldt I. Prevalence of Streptococcus sanguis andStreptococcus mutans in the mouth of persons wearing full-dentures. Arch OralBiol,1969,14:243–249
[9] Chastre J. Evolving problems with resistant pathogens. Clin Microbiol Infect,2008,14(Suppl3):3-14
[10]Choi BK, Kim KY, Yoo YJ, Oh SJ, Choi JH, Kim CY. In vitro antimicrobialactivity of a chitooligosaccharide mixture against Actinobacillusactinomycetemcomitans and Streptococcus mutans. Int J Antimicrob Agents,2001,18(6):553-557
[11]Hancock RE, Patrzykat A. Clinical development of cationic antimicrobialpeptides: from natural to novel antibiotics. Curr Drug Targets Infect Disord,2002,2:79-83
[12]Riley MA, Wertz JE. Bacteriocins: evolution, ecology, and application. AnnuRev Microbiol,2002,56:117-137
[13]Ajdic D, McShan WM, McLaughlin RE, Savic G, Chang J, Carson MB,Primeaux C, Tian R, Kenton S, Jia H, Lin S, Qian Y, Li S, Zhu H, Najar F, Lai H,White J, Roe BA, Ferretti JJ. Genome sequence of Streptococcus mutansUA159--a cariogenic dental pathogen. Proc Natl Acad Sci U.S.A.2002,99(22):14434-14439
[14]Brown ED, Vivas EI, Walsh CT, Kolter R. MurA (MurZ), the enzyme thatcatalyzes the first committed step in peptidoglycan biosynthesis, is essential inEscherichia coli. J Bacteriol,1995,177:4194–4197
[15]da Silva BR, de Freitas VA, Nascimento-Neto LG, Carneiro VA, Arruda FV, deAguiar AS, Cavada BS, Teixeira EH. Antimicrobial peptide control ofpathogenic microorganisms of the oral cavity: a review of the literature.Peptides,2012,36(2):315-21
[1] Hancock RE, Patrzykat A. Clinical development of cationic antimicrobialpeptides: from natural to novel antibiotics. Curr Drug Targets Infect Disord,2002,2:79-83
[2] Riley MA, Wertz JE. Bacteriocins: evolution, ecology, and application.Annu Rev Microbiol,2002,56:117-137
[3] Merrifield EL, Mitchell S, Ubach J, Boman HG, Andreu D, Merrifield RB.D-enantiomers of15-residue cecropin A-melittin hybrids. Int J Pept ProteinRes,1995,46:214-220
[4] Oren Z, Ramesh J, Avrahami D, Suryaprakash N, Shai Y, Jelinek R. Modeof membrane interaction and structure in micelles of a short a-helical lyticpeptide and its diastereomer determined by NMR, FTIR, and fluorescencespectroscopy. Eur. J. Biochem,2002,269:3869-3880
[5] Wu JM, Wei SY, Chen HL, Weng KY, Cheng HT, Cheng JW. Solutionstructure of a novel D-β-naphthylalanine substituted peptide with potentialantibacterial and antifungal activities. Biopolymers,2007,88:738-745
[6] Clinical and Laboratory Standards institute. Performance standards forantimicrobial susceptibility testing: fifteenth informational supplementM100-S15. CLSI, Wanye PA, USA2005
[7] Liu N, Chen XG, Park HJ, Liu CG, Liu CS, Meng XH, Yu LJ. Effect ofMW and concentration of chitosan on antibacterial activity of Escherichiacoli. Carbohydrate Polymer,2006,64:60–65
[8] Liu H, Du Y, Wang X, Sun L. Chitosan kills bacteria through cellmembrane damage. Int J Food Microbiol,2004,95(2):147-155
[9] Stepanovic S, Cirkovic I, Ranin L, Svabic-Vlahovic M. Biofilm formationby Salmonella spp. and Listeria monocytogenes on plastic surface. LettAppl Microbiol,2004,38:428-432
[10]Russell RR. How has genomics altered our view of caries microbiology?Caries Res,2008,42:319-327
[11]Wilson M. Susceptibility of oral bacterial biofilms to antimicrobial agents. JMed Microbiol,1996,44:79-87
[12]Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA,Sahasrabudhe A, Dewhirst FE. Bacterial diversity in human subgingivalplaque. J Bacteriol,2001,183(12):3770-3783
[13]Marsh PD. Are dental diseases examples of ecological catastrophes?Microbiology,2003,149:279-294
[14]Cintas LM. Bacteriocins of Lactic Acid Bacteria. Food Scie Tech Int,2001,7(4):281-305
[15]Liu Y, Wang L, Zhou X, Hu S, Zhang S, Wu H. Effect of the antimicrobialdecapeptide KSL on the growth of oral pathogens and Streptococcusmutans biofilm. Int J Antimicrob Agents,2011,37(1):33-8.
[16]Wei GX, Campagna AN, Bobek LA. Effect of MUC7peptides on thegrowth of bacteria and on Streptococcus mutans biofilm. J AntimicrobChemother,2006,57:1100-1109
[17]Raj PA, Antonyraj KJ, Karunakaran T. Large-scale synthesis and functionalelements for the antimicrobial activity of defensins. Biochem J,2000,347(3):633-41
[18]Beckloff N, Laube D, Castro T, Furgang D, Park S, Perlin D, Clements D,Tang HZ, Scott RW, Tew GN, Diamond G. Activity of an antimicrobialpeptide mimetic against planktonic and biofilm culture of oral pathogens.Antimicrob Agents Chemother,2007,51(11):4125-32
[19]Zasloff M. Antimicrobial peptides of multicellular organisms. Nature,2002,415:389-395
[20]Cabrera JE, Caglier C, Quan S, Squires CL, Jin DJ. Active Transcription ofrRNA Operons Condenses the Nucleoid in Escherichia coli: Examining theEffect of Transcription on Nucleoid Structure in the Absence of Transertion.J. Bacteriol,2009,191:4180-4185
[21]Shemesh M, Tam A, Steinberg D. Differential gene expression profiling ofStreptococcus mutans cultured under biofilm and planktonic conditions.Microbiology,2007,153:1307-1317
[22]Chitnis SN and Prasad KS.Seminalplasmin, an antimicrobial protein frombovine seminal plasma, inhibits peptidoglycan synthesis in Escherichia coli.FEMS Microbiol Lett,1990,60:281-284.
[23]Wang Y, Lee SM, Dykes GA. Potential mechanisms for the effects of teaextracts on the attachment, biofilm formation and cell size of Streptococcusmutans. Biofouling,2013,29(3):307-18.
[1] Park JT, Neidhardt R, Curtiss III, Ingraham JL, ECC Lin, Low KB, Magasanik B. Escherichia coli and Salmonella: cellular and molecular biology,2nd ed.1996.vol.1. ASM Press, Washington, D.C.
[2] Barreteau H, Kovac A, Boniface A, Sova M, Gobec S, Blanot D. Cytoplasmic steps of peptidoglycan biosynthesis. FEMS Microbiol Review,2008,(32):168-207
[3] Zhang WL, Jones VC, Scherman MS, Mahapatra S, Crick D, BhamidiS, Xin Y, McNeil MR, Ma YF. Expression, essentiality, and a microtiter plate assay for mycobacterial GlmU, the bifunctional glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase. Int J Biochem Cell Biol,2008,40(11):2560-2571
[4] Clifton E, Barry, Dean C, Crick, Michael R. McNeil. Targeting the formation of the cell wall core of M. tuberculosis. Infectious Disorders Drug Targets,2007,7:1-21
[5] Brown ED, Vivas EI, Walsh CT, Kolter R. MurA (MurZ), the enzymethat catalyzes the first committed step in peptidoglycan biosynthesis, isessential in Escherichia coli. J Bacteriol,1995,177:4194–4197
[6] Marquardt JL, Siegele DA, Kolter R, Walsh CT. Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase. J Bacteriol,1992,174:5748-5752
[7] Wanke C, Falchetto R, Amrhein N. The UDP-N-acetylglucosamine1-carboxyvinyl-transferase of Enterobacter cloacae. Molecular cloning, sequencing of the gene and overexpression of the enzyme. FEBS Lett,1992,301:271-276
[8] Benson TE, Marquardt JL, Marquardt AC, Etzkorn FA, Walsh CT. Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase. Biochemistry,1993,32:2024-2030
[9] Benson TE, Harris MS, Choi GH, Cialdella JI, Herberg JT, Martin JP,Baldwin ET. A structural variation for MurB--X-ray crystal structure ofStaphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase(MurB).Biochemistry,2001,40:2340-2350
[10]钟启平.口腔微生物学,第一版,北京,人民卫生出版社,2009;165-173
[11]Kumar S, Parvathi A, Hernandez RL, Cadle KM, Varela MF. Identification of a novel UDP-N-acetylglucosamine enolpyruvyl transferase (MurA)from Vibrio fischeri that confers high fosfomycin resistance in Escherichia coli. Arch Microbiol,2009,191:425-429
[12]Gunetileke KG, Anwar RA. Biosynthesis of uridine diphospho-N-acetylmuramic acid II. Purification and properties of pyruvate-uridine diphospho-N-acetylglucosamine transferase and characterization of uridine diphospho-N-acetylenopyruvylglucosamine. J Biol Chem,1968,243:5770-5778
[13]Mengin-Lecreulx D, Flouret B, van Heijenoort J. Pool levels of UDP N-acetylglucosamine and UDP N-acetylglucosamine-enolpyruvate in Escherichia coli and correlation with peptidoglycan synthesis. J Bacteriol,1983,154:1284-1290
[14]Eschenburg S, Priestman M, Schonbruns E. Evidence that the fosfomycin target Cys115in UDP-N-acetylglucosamine enolpyruvyl transferase(MurA) is essential for product release. J Biol Chem,2005,280:3757-3763
[15]DeSmet KA, Kempsell KE, Gallagher A, Duncan K, Young DB. Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis. Microbiology,1999,145:3177-3184
[16]McCoy AJ, Sandlin RC, Maurulli AT. In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involve in peptidoglycan synthesis and fosfomycin resistance. JBacteriol,2003,185:1218-1228
[17]Russell RR. How has genomics altered our view of caries microbiology?Caries Res,2008,42:319-327
[18]Lemos JA, Burne RA. A model of efficiency--stress tolerance by Streptococcus mutans. Microbiology,2008,154:3247-3255
[19]Blake KL, O'Neill AJ, Mengin-Lecreulx D, Henderson PJ, Bostock JM,Dunsmore CJ, Simmons KJ, Fishwick CW, Leeds JA, Chopra I. Thenature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors. Mol Microbiol,2009,72(2):335-43
[20]Han SG, Lee WK, Jin BS, Lee KI, Lee HH, Yu YG. Identification ofNovel Irreversible Inhibitors of UDP-N-Acetylglucosamine EnolpyruvylTransferase (MurA) from Haemophilus influenzae. J Microbiol Biotechnol,2013,23(3):329-34.
[1] Beutler B. Innate immunity--an overview. Molecular Immunology,2004,40:845–859
[2] Hancock RE and Sahl HG. Antimicrobial and host-defense peptides as newanti-infective therapeutic strategies. Nature Biotechnology,2006,24:1551–1557
[3] Gordon YJ, Romano ski EG, McDermott AM. A review of antimicrobialpeptides and their therapeutic potential as anti-infective drugs. Current EyeResearch,2005,30:505–515
[4] McPhee JB, Hancock RE. Function and therapeutic potential of host defensepeptides. Journal of Peptide Science,2005,11:677–687
[5] Yeaman MR, Yount NY. Unifying themes in host defense effecterpolypeptides. Nature Reviews Microbiology,2007,5:727–740
[6] Linde A, Ross CR, Davis EG, Dib L, Blecha F, Melgarejo T. Innate immunityand host defense peptides in veterinary medicine. Journal of VeterinaryInternal Medicine,2008,22:247–265
[7] Sang Y and Blecha F. Porcine host defense peptides--expanding repertoireand functions. Developmental and Comparative Immunology,2009,33(3):334-43
[8] Boman HG. Antibacterial peptides--basic facts and emerging concepts.Journal of Internal Medicine,2003,254:197–215
[9] Cotter PD, Hill C, Ross RP. Bacteriocins--developing innate immunity forfood. Nature Reviews Microbiology,2005,3:777–788
[10]Willey JM, Vander Dunk WA. Antibiotics--peptides of diverse structure andfunction. Annual Review of Microbiology,2007,61:477–501
[11]Hammami R, Zouhir A, Ben Hamida J, Fliss I. BACTIBASE--aweb-accessible database for bacteriocin characterization.BMC Microbiology,2007,7:89
[12]De Vuyst L, Leroy F. Bacteriocins from lactic acid bacteria--production,purification, and food applications.Journal of Molecular Microbiology andBiotechnology,2007,13:194–199
[13]Sit CS, Vederas JC. Approaches to the discovery of new antibacterial agentsbased on bacteriocins. Biochemistry and Cell Biology,2008,86:116–123
[14]Dufour A, Hindre T, Haras D, Le Pennec JP. The biology of antibiotics fromthe lactic in481group is coming of age. FEMS Microbiology Reviews,2007,31:134–167
[15]Crispie F, Twomey D, Flynn J, Hill C, Ross P, Meaney W. The antibioticlactic in3147produced in a milk based medium improves the efficacy of abismuth-based teat seal in cattle deliberately infected with Staphylococcusaurous.Journal of Dairy Research,2005,72:159–167
[16]Gardiner GE, Rea MC, O’Riordan B, O’Connor P, Morgan SM, Lawlor PG,Lynch PB, Cronin M, Ross RP, Hill C. Fate of the two-component antibioticlactic in3147in the gastrointestinal tract. Applied and EnvironmentalMicrobiology,2007,73:7103–7109
[17]Kruszewska D, Sahl HG, Bierbaum G, Pag U, Hynes SO, Ljungh A.Mersacidin eradicates methicillin-resistant Staphylococcus aureus (MRSA) ina mouse rhinitis model.Journal of Antimicrobial Chemotherapy,2004,54:648–653
[18]Duquesne S, Destoumieux Garzon D, Peduzzi J, Rebuffat S. Microclines,gene-encoded antibacterial peptides from enter bacteria. Natural ProductReports,2007,24:708–734
[19]Nes IF, Diep DB, Holo H. Bacteriocin diversity in Streptococcus andEnterococcus. Journal of Bacteriology,2007,189:1189–1198
[20]Rossi LM, Rangasamy P, Zhang J, Qiu XQ, Wu GY.Research advances in thedevelopment of peptide antibiotics. Journal of Pharmaceutical Sciences,2008,97:1060–1070
[21]Duclohier H. Peptaibiotics and peptaibols--an alternative to classicalantibiotics. Chemistry and Biodiversity,2007,4:1023–1026
[22]Leitgeb B, Szekeres A, Manczinger L, Vagvolgyi C, Kredics L. The history ofalamethicin--a review of the most extensively studied peptaibol. Chemistryand Biodiversity,2007,4:1027–1051
[23]Zhu S. Discovery of six families of fungal defending-like peptides providesinsights into origin and evolution of the C Salphabeta defenses. MolecularImmunology,2008,45:828–838
[24]Mygind PH, Fischer RL, Schnorr KM, Hansen MT, Sonksen CP, Ludvigsen S,Ravento′s D, Buskov S, Christensen B, De Maria L, Taboureau O, Yaver D,Elvig-J rgensen SG, S rensen MV, Christensen BE, Kjaerulff S,Frimodt-Moller N, Lehrer RI, Zasloff M, Kristensen HH. Plectasin is apeptide antibiotic with therapeutic potential from asaprophytic fungus. Nature,2005,437:975–980
[25]Silverstein KA, Graham MA, Paape TD, VandenBosch KA. Genomeorganization of more than300defensinlike genes in Arabidopsis. PlantPhysiology,2005,138:600–610
[26]Thevissen K, Kristensen HH, Thomma BP, Cammue BP, Francois IE.Therapeutic potential of antifungal plant and insect defensins. Drug DiscoveryToday,2007,12:966–971
[27]Zhang L, Dhillon P, Yan H, Farmer S, and Hancock RE. Interactions ofbacterial cationic peptide antibiotics with outer and cytoplasmic membranesof Pseudomonas aeruginosa. Antimicrob Agents Chemother,2000,44:3317–3321.
[28]Ireland DC, Wang CK, Wilson JA, Gustafson KR, Craik DJ. Cyclotides asnatural anti-HIV agents. Biopolymers,2008,90:51–60
[29]Barbeta BL, Marshall AT, Gillon AD, Craik DJ, Anderson MA. Plantcyclotides disrupt epithelial cells in the midgut of lepidopteran larvae.Proceedings of the National Academy of Science, USA,2008,105:1221–1225
[30]Colgrave ML, Kotze AC, Huang YH, O’Grady J, Simonsen SM, Craik DJ.Cyclotides--natural, circular plant peptides that possess significant activityagainst gastrointestinal nematode parasites of sheep. Biochemistry,2008,47:5581–5589
[31]Whittington CM, Papenfuss AT, Bansal P, Torres AM, Wong ES, Deakin JE,Graves T, Alsop A, Schatzkamer K, Kremitzki C, Ponting CP, Temple-SmithP, Warren WC, Kuchel PW, Belov K. Defensins and the convergent evolutionof platypus and reptile venom genes. Genome Research,2008,18:986–994
[32]Warren WC, Hillier LW, Marshall Graves JA, Birney E, Ponting CP,Gru¨tzner F, Belov K. Genome analysis of the platypus reveals uniquesignatures of evolution. Nature,2008,453:175–183
[33]Ganz T. Defensins--antimicrobial peptides of innate immunity. NatureReviews Immunology,2003,3:710–720
[34]Selsted ME and Ouellette AJ. Mammalian defensins in the antimicrobialimmune response. Nature Immunology,2005,6:551–557
[35]Lehrer RI. Multispecific myeloid defensins. Current Opinion in Hematology,2007,14:16–21
[36]Lynn DJ, Bradley DG. Discovery of alpha-defensins in basal mammals.Developmental and Comparative Immunology,2007,31:963–967
[37]Brogden KA. Antimicrobial peptides--pore formers or metabolic inhibitors inbacteria? Nature Reviews Microbiology,2005,3:238–250
[38]Zaiou M. Multifunctional antimicrobial peptides--therapeutic targets inseveral human diseases. Journal of Molecular Medicine,2007,85:317–329
[39]Kang JH, Lee MS. Characterization of a bacteriocin produced byEnterococcus faecium GM-1isolated from an infant. Journal of AppliedMicrobiology,2005,98:1169–1176
[40]Svetoch EA, Eruslanov BV, Perelygin VV, Mitsevich EV, Mitsevich IP,Borzenkov VN, Levchuk VP, Svetoch OE, Kovalev YN, Stepanshin YG,Siragusa GR, Seal BS, Stern NJ. Diverse antimicrobial killing byEnterococcus faecium E50-52bacteriocin. Journal of Agricultural and FoodChemistry,2008,56:1942–1948
[41]Wiedemann I, Benz R, Sahl HG. Lipid II-mediated pore formation by thepeptide antibiotic Nisin--a black lipid membrane study. J Bacteriol,2004,186：3259–3261
[42]van Kraa CE, Breukink HS, Rollema R J, Siezen RJ, Demel RA, De Kruijff B,Kuipers OP．Influence of charge differences in the C-terminal part of Nisin onantimicrobial activity and signaling capacity. Eur J Biochem,1997,247：l14–120
[43]Huang HW. Molecular mechanism of antimicrobial peptides--the origin ofcooperativity. Biochimica et Biophysica Acta,2006,1758:1292–1302
[44]Jang H, Ma B, Woolf TB, Nussinov R. Interaction of protegrin-1with lipidbilayers--membrane thinning effect.Biophysical,2006,91:2848–2859
[45]Mani R, Cady SD, Tang M, Waring AJ, Lehrer RI, Hong M.Membrane-dependent oligomeric structure and pore formation of abeta-hairpin antimicrobial peptide in lipid bilayers from solid-state NMR.Proceedings of the National Academy of Sciences, USA,2006,103:16242–16247
[46]Murakami M, Lopez-Garcia B, Braff M, Dorschner RA, Gallo RL.Postsecretory processing generates multiple cathelicidins for enhanced topicalantimicrobial defense. Journal of Immunology,2004,172:3070–3077
[47]Draper LA, Ross RP, Hill C, Cotter PD. Lantibiotic immunity, CurrentProtein and Peptide. Science,2008,9:39–49
[48]Scott MG, Dullaghan E, Mookherjee N, Glavas N, Waldbrook M, ThompsonA, Wang A, Lee K, Doria S, Hamill P, Yu JJ, Li Y, Donini O, Guarna MM,Finlay BB, North JR, Hancock RE. An anti-infective peptide that selectivelymodulates the innate immune response. Nature Biotechnology,2007,25:465–472
[49]Gunn JS. The Salmonella PmrAB regulon--lipopolysaccharide modifications,antimicrobial peptide resistance and more. Trends in Microbiology,2008,16:284–290
[50]Kraus D, Peschel A. Staphylococcus aureus evasion of innate antimicrobialdefense. Future Microbiology,2008,3:437–451
[51]Andres E, Dimarcq JL. Clinical development of antimicrobial peptidesInternational. Journal of Antimicrobial Agents,2005,25:448–449
[52]Bansal PS, Torres AM, Crossett B, Wong KK, Koh JM, Geraghty DP,Vandenberg JI, Kuchel PW. Substrate specificity of platypus venomL-to-D-peptide isomerase. Journal of Biological Chemistry,2008,283:8969–8975
[53]Jenssen H, Fjell CD, Cherkasov A, Hancock RE. QSAR modeling andcomputer-aided design of antimicrobial peptides. Journal of PeptideScience,2008,14:110–114
[54]Hyvonen P, Suojala L, Orro T, Haaranen J, Simola O, R ntved C, Pyorala S.Transgenic cows that produce recombinant human lactoferrin in milk are notprotected from experimental Escherichia coli intramammary infection.Infection and Immunity,2006,74:6206–6212
[55]Han ZS, Li QW, Zhang ZY, Yu YS, Xiao B, Wu SY, Jiang ZL, Zhao Hw,Zhao R, Li J. Adenoviral vector mediates high expression levels of humanlactoferrin in the milk of rabbits. Journal of Microbiology and Biotechnology,2008,18:153–159
[56]Zhang J, Li L, Cai Y, Xu X, Chen J, Wu Y, Yu H, Yu G, Liu S, Zhang A,Chen J, Cheng G. Expression of active recombinant human lactoferrin in themilk of transgenic goats. Protein Expression and Purification,2008,57:127–135
[57]Wu SC, Chen HL, Yen CC, Kuo MF, Yang TS, Wang SR, Weng CN, et al.Recombinant porcine lactoferrin expressed in the milk of transgenic miceenhances offspring growth performance. Journal of Agricultural and FoodChemistry,2007,55:4670–4677
[58]Chen HL, Wang LC, Chang CH, Yen CC, Cheng WT, Wu SC, Hung CM,Kuo MF, Chen CM. Recombinant porcine lactoferrin expressed in the milk oftransgenic mice protects neonatal mice from a lethal challenge withenterovirus type71. Vaccine,2008,26:891–898
[59]Humphrey BD, Huang N, Klasing KC. Rice expressing lactoferrin andlysozyme has antibiotic-like properties when fed to chicks. Journal ofNutrition,2002,132:1214–1218
[60]Salzman NH, Ghosh D, Huttner KM, Paterson Y, Bevins CL. Protectionagainst enteric salmonellosis in transgenic mice expressing a human intestinaldefensin.Nature,2003,422:522–526
[61]Lee PH, Ohtake T, Zaiou M, Murakami M, Rudisill JA, Lin KH, Gallo RL.Expression of an additional cathelicidin antimicrobial peptide protects againstbacterial skin infection.Proceedings of the National Academy of Sciences,USA2005,102:3750–3755
[62]Cheung QC, Turner PV, Song C, Wu D, Cai HY, MacInnes JI, Li J. Enhancedresistance to bacterial infection in protegrin-1transgenic mice. AntimicrobialAgents and Chemotherapy,2008,52:1812–1819
[63]Yamaguchi Y, Nagase T, Tomita T, Nakamura K, Fukuhara S, Amano T,Yamamoto H, Ide Y, Suzuki M, Teramoto S, Asano T, Kangawa K, NakagataN, Ouchi Y, Kurihara H. Beta-defensin overexpression induces progressivemuscle degeneration in mice. American Journal of Physiology–CellPhysiology,2007,292: C2141–C2149
[64]Peters BM, Shirtliff ME, Jabra-Rizk MA. Antimicrobial Peptides:PrimevalMolecules or Future Drugs? PLoS Pathog2010;6(10): e1001067.doi:10.1371/journal.ppat.1001067
[65]Guaní-Guerra E, Santos-Mendoza T, Lugo-Reyes SO, Teran LM.Antimicrobial peptides--general overview and clinical implications in humanhealth and disease. Clin Immunol,2010,135(1):1-11
[66] Chitnis SN and Prasad KS.Seminalplasmin, an antimicrobial protein frombovine seminal plasma, inhibits peptidoglycan synthesis in Escherichiacoli.FEMS Microbiol Lett,1990,60:281–284.
[67]Xiong YQ, Bayer AS, and Yeaman MR.Inhibition of intracellularmacromolecularsynthesis in Staphylococcus aureus by thrombin-inducedplatelet microbicidalproteins. J Infect Dis,2002,186:668–677.
[68]Park CB, Kim HS, and Kim SC. Mechanism of action of theantimicrobialpeptide buforin II: buforin II kills microorganisms by penetratingthe cell membraneand inhibiting cellular functions. Biochem Biophys ResCommun,1998,244:253–257.
[69]del Castillo FJ, del Castillo I, and Moreno F. Construction andcharacterization of mutations at codon751of the Escherichia coli gyrB genethat confer resistance to the antimicrobial peptide microcin B17and alter theactivity of DNA gyrase. J Bacteriol,2001,183:2137–2140.