【作者】 邱家章；

【导师】 邓旭明；

【作者基本信息】 吉林大学 ， 基础兽医学， 2012， 博士

【摘要】 金黄色葡萄球菌（Staphylococcus aureus,简称为金葡菌）是常见的革兰氏阳性致病菌，是极其重要的人兽共患病原体。金葡菌所导致的疾病范围非常广，从普通的皮肤软组织感染到危及生命的肺炎、心内膜炎和毒素休克综合症等，具有非常高的发病率和死亡率。在兽医临床上，金葡菌是奶牛和羊乳房炎的主要病原，造成极大的经济损失以及公共卫生问题。金葡菌的耐药性非常严重，几乎对所有的抗生素都产生了耐药性，尤其是MRSA菌株的传播和蔓延，已经成为人类及动物健康的重要威胁。90%的MRSA菌株具有多药耐药特征，表现为对多种抗菌药物如β-内酰胺类、喹诺酮类、大环内酯类和四环素类等多种抗菌药物同时耐药。长期以来，糖肽类抗生素万古霉素被认为是治疗MRSA感染的首选药物，但随着万古霉素对金葡菌敏感性的降低甚至耐药，使得临床上对MRSA感染面临一种无药可用的局面。因此，临床上迫切需要开发新的抗金葡菌感染药物。随着人们对金葡菌致病机制和信号传导通路研究的深入，有一些新的抗感染策略获得了广泛的关注。众所周知，金葡菌分泌大量的细胞表面蛋白和外毒素，参与细菌的致病过程，因此，以毒力因子为靶标的抗感染药物的开发成为非常有前景的控制金葡菌感染的策略。α-溶血素是金葡菌在对数生长后期分泌的主要外毒素之一，为33.2kDa的可溶性蛋白，包含293个氨基酸残基，可导致多种细胞的裂解死亡，包括红细胞、淋巴细胞、上皮细胞、巨噬细胞和单核细胞等。α-溶血素需要在敏感细胞的细胞膜表面自组装形成七聚体，引起细胞内容物的渗漏，进而导致细胞的死亡。α-溶血素在金葡菌感染中所起的作用已研究的非常透彻，缺乏α-溶血素表达的金葡菌菌株在多种实验动物疾病模型中表现致病性显著降低，如乳房炎、腹膜炎、角膜炎和肺炎等。基于α-溶血素的细胞裂解机制及其在疾病过程的关键作用，α-溶血素有望成为抗金葡菌感染药物开发的重要靶标。天然化合物是新药发现的重要资源，而抗毒力药物的研发依赖于具有抗毒力特性的半合成或天然化合物的发现。本研究通过溶血试验、western-blot和荧光定量PCR试验，从表型功能、蛋白水平和基因水平三个层面上研究了22种天然化合物对金葡菌α-溶血素的影响。发现异土木香内酯、甘草查尔酮A、杜鹃素、辣椒碱、麝香草酚、丁香酚、木犀草素、白杨黄素和薄荷脑通过抑制α-溶血素编码基因hla的转录，从而降低α-溶血素的表达，进而抑制其溶血活性。α-溶血素的表达受到由多个调控子组成的调控网络的控制，其中Agr二元调控系统处于核心的地位。为此，本研究进一步通过荧光定量PCR证明了上述九种天然化合物均可显著地抑制agrA的转录，提示这些化合物抑制α-溶血素的表达可能部分地依赖于Agr二元调控系统，但是不排除其它调控子也受到这些化合物的影响。黄芩苷是传统中药黄芩的主要有效成分，属黄酮类化合物，具有非常广泛的药理活性，包括抗炎、抗病毒、抗肿瘤、抗过敏和解热等作用。与上述9种化合物不同，黄芩苷作用于金葡菌后，能够剂量依赖性地抑制金葡菌培养物上清对兔红细胞的溶血活性，但western-blot结果显示α-溶血素的表达量并没有变化，提示黄芩苷可能直接作用α-溶血素结构本身，随后应用纯化的α-溶血素与黄芩苷作用后，其溶血作用显著降低，从而证明了这种可能性。本研究应用理论化学的方法，应用分子对接和分子动力学模拟技术，以阐明黄芩苷抑制α-溶血素的机制。通过同源模建获得α-溶血素的初始结构，应用AutoDock4.0对α-溶血素与黄芩苷进行对接，能量的算法使用拉马克遗传算法（LGA），全面搜索最优化构象。随后应用GROMACS4.5.3程序包对分子对接的结果进行分子动力学模拟，应用MM-GBSA方法计算结合自由能。结果表明，黄芩苷可结合与α-溶血素结构的“三角区”，Tyr148、Pro151和Phe153为其结合的主要残基，结合方式主要是通过疏水作用；黄芩苷与α-溶血素的结合使α-溶血素“三角区”两边的构象变化受到束缚，无法进行自由伸缩，从而不能形成七聚体。本研究应用定点残基突变和荧光淬灭试验验证黄芩苷与α-溶血素的结合位点。通过将α-溶血素氨基酸序列中的Tyr148、Pro151和Phe153突变为丙氨酸（Ala），表达和纯化野生型α-溶血素及突变体α-溶血素，应用荧光淬灭试验测定结合常数，结果表明，突变后的α-溶血素与黄芩苷的结合明显降低，结合常数大小为野生型> F153A> P151A> Y148A。通过脱氧胆酸诱导的寡聚化和SDS-PAGE验证了黄芩苷对α-溶血素七聚体形成的影响。结果显示，黄芩苷作用后α-溶血素七聚体的量显著降低，该作用呈现剂量依赖性。本研究将金葡菌与肺泡上皮细胞A549共培养体系中加入黄芩苷，进行激光共聚焦观察和LDH含量测定，结果表明黄芩苷能够显著保护由α-溶血素介导的A549细胞的损伤。提示黄芩苷对金葡菌肺炎的潜在治疗作用。本研究进一步应用高效液相色谱法考察了黄芩苷在小鼠体内的药代动力学特征。小鼠分别皮下注射25、50和100mg/kg黄芩苷后，Cmax分别为4.5、14.81和24.74μg/ml，半衰期为1.29h，符合一级吸收的开放一室模型。通过建立小鼠金葡菌肺炎模型，根据黄芩苷在小鼠体内的药动学参数设计给药方案，考察黄芩苷对金葡菌肺炎的治疗作用。结果显示，黄芩苷治疗后显著降低金葡菌肺炎小鼠的死亡率，降低小鼠肺脏金葡菌的定殖数量；肺脏湿重/干重比降低，提示肺水肿症状的减轻；组织病理学显示黄芩苷治疗后显示肺组织充血减轻，肺泡腔炎性细胞浸润明显减少，肺泡结构相对完整，提示肺部炎症的缓解。肺泡灌洗液分析试验结果表明，黄芩苷治疗显著抑制感染小鼠肺部嗜中性粒细胞的聚集，主要炎症因子TNF-α、IL-1β和IL-6的含量降低。综上所述，黄芩苷体外可通过阻碍金葡菌α-溶血素七聚体的自组装，从而抑制其细胞溶解活性；黄芩苷可保护α-溶血素介导的A549细胞的损伤；实验治疗学显示黄芩苷可有效地治疗小鼠金葡菌肺炎。本研究为阐明黄芩苷抗感染的机制以及创新性抗感染药物的研发奠定了坚实的基础。更多还原

【Abstract】 Staphylococcus aureus is a gram-positive zoonotic pathogen, causing a broadrange of diseases, ranging from relative minor skin and soft tissue infections tolife-threatening pneumonia, endocarditis, meningitis, septicemia and toxic shocksyndrome. In the veterinary clinic, S. aurues is the major pathogen that causedmastitis of cows and sheep, resulting in great economic losses and public health issues.The antibiotic resistance of S. aurues is pretty serious. Actually, it has been resistant toall clinically used antibiotics. Particularly, the dissemination and wide spread ofmethicillin-resistant S. aurues (MRSA) isolates have become a major threat to thehuman and animal health.90%of MRSA isolates have multi-drug resistant featurethat are simultaneously resistant to many antimicrobial agents, such as β-lactams,quinolones, macrolides and tetracyclines. The glycopeptides antibiotic vancomycin ispreviously considered to be the preferred drug for the treatment of MRSA infections.However, the emergence of vancomycin-insensitive S. aurues (GISA) andvancomycin-resistant S. aurues strains (VRSA) has made it difficult to controlstaphylococcal infections. Consequently, there is an urgent need to develop moreeffective antimicrobial agents or alternative therapeutic strategies.With increasing understanding of bacterial pathogenesis and intercellularcommunication has revealed many potential strategies to develop novel drugs for thetreatment of S. aurues-mediated diseases. S. aurues produces a number of virulencefactors, including cell surface proteins and exotoxins, which participated inpathogenesis. Presently, targeting virulence factors has become a promising strategyfor the treatment of S. aurues illnesses. α-hemolysin (Hla) is one of the majorexotoxins of S. aurues that is produced in the post-exponential growth phase. It issecreted as a33.2kDa water-soluble extracellular protein which contains293aminoacids. A wide range of mammalian cells, including erythrocytes, monocytes,lymphocytes, macrophages and epithelial cells, are influenced by Hla. Hla is a self-assembling, channel-forming toxin. Upon binding to the membrane of asusceptible cell, the membrane-bound monomer oligomerizes to form a232.4-kDamembrane-inserted heptamer. The heptameric pore leads to leakage of ions, water,and low molecular weight molecules out of and into the cell, and ultimately causingcell lysis and death. The roles of Hla in S. aurues infections have beenwell-investigated. Strains lacking Hla displayed less virulent in animal models ofdiseases, such as mastitis, peritonitis, keratitis and pneumonia. Based on the cell lyticmechanism and the key roles of Hla in S. aurues infections, Hla could be an importanttarget for the development of anti-S. aurues drugs.Natural compounds are important resources for drug discovery, while thedevelopment of anti-virulence drug relies on newly discovered synthetic or naturalsmall organic compounds that possess anti-virulence characteristics. In the study, weapplied hemolysis assay, western-blot and real-time RT-PCR to investigate theinfluence of22natural compounds on the production of Hla. The results indicated thatisoalantolactone, licochanlcone A, farrerol, capsaicin, thymol, eugenol, luteolin,chrysin and menthol could inhibit the transcription of hla gene (encoding Hla),decrease the production of Hla, and thereby inhibiting the hemolytic activity. Theexpression of Hla is controlled by a network of regulators, among which the Agrtwo-component system is the most important and well-characterized one. The data ofreal-time RT-PCR showed that the transcription of agrA was significantly inhibited bythese compounds, suggesting that the decreased production of Hla may partiallydepend on the inhibition of Agr regulator, although other regulators may also beinfluenced. Baicalin is the major component of Scutellaria baicalensis Georgi (HuangQin), which belongs to the flavonoids. It has various biological effects, includinganti-inflammatory, anti-viral, anti-tumor, anti-allergy and anti-pyretic characteristics.Treatment with baicalin could also inhibit the hemolytic activities of S. aurues culturesupernatants; however, the western-blot results showed that the expression of Hlawere not affected, suggesting that baicalin may directly inhibit Hla. Subsequently, wedemonstrated the possibility by hemolysis assay using purified Hla.Further, we used the theoretical chemistry methods to investigate the mode ofaction that baicalin inactivates Hla. Molecular docking and molecular dynamics (MD) simulations were employed. The initial structure of Hla was obtained from thehomology modeling. A standard docking procedure for Hla and baicalin wasperformed with AutoDock4.0, while the Lamarckian genetic algorithm (LGA) wasapplied in the docking calculations to obtain optimal conformation. MD simulationswere performed with the Gromacs4.5.3software package to analyze the moleculardocking data. The binding free energies were calculated using MM-GBSA approachsupplied by Amber10package. The results showed that baicalin could bind to the“triangle region” of the Hla monomer, while Tyr148, Pro151and Phe153were themajor binding sites. The hydrophobic interaction was the principal binding mode; thebinding of bacicalin restrained the conformational change of “triangle region”,thereby inhibiting the self-assembly of the heptameric transmembrane pore.Site-directed mutagenesis and fluorescence quenching assays were applied toverify the binding sites of baicalin on Hla. The Tyr148, Pro151and Phe153in Hlawere mutated to Alanine (Ala). We constructed plasmids encoding wild-type(WT)-Hla, Y148A-Hla, P151A-Hla and F153A-Hla. Further, WT-Hla, Y148A-Hla,P151A-Hla and F153A-Hla were expressed and purified. The purified proteins wereused for determination of fluorescence quenching constants. The results showed thatmutations of Hla led to reduction of baicalin binding. The binding constants of theinteraction between baicalin and Hla decrease in the following order: WT-Hla>F153A-Hla> P151A-Hla> Y148A-Hla. Moreover, we used thedeoxycholate-induced oligomerization assay and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to assess the influence ofbaicalin on the formation of Hla heptamer. Apparently, the addition of baicalinresulted in concentration-dependent reduction of Hla heptamer formation.Alveolar epithelial cells (A549) were co-cultured with S. aurues. Followingtreatment with baicalin, cell viability was measured either using the confocal laserscanning microscope or by measuring LDH. The data indicated that baicalin couldprotect Hla mediated injury of A549cells, suggesting the potential therapeutic effectof baicalin in staphylococcal pneumonia.The pharmacokinetic characteristics of baicalin in mice were determined by highperformance liquid chromatography (HPLC). The maximum concentrations of baicalin in plasma (Cmax) were4.5,14.81and24.74μg/ml for single subcutaneousdoses of25,50and100mg/kg, respectively. The half-time (t1/2) is about1.29h. Thepharmacokinetics of baicalin conformed to one-compartment open model with firstorder absorption.The mouse model of S. aurues pneumonia was established to assess the in vivoperformance of baicalin, and the dosage regimen was designed according to thepharmacokinetic characteristics of baicalin in mice. The results showed that treatedmice with baicalin significantly decreased the mortality and the colony forming units(CFUs) in the lung tissues. The lung wet-dry ratio was also reduced, indicating thatthe pulmonary edema was alleviated. Histopathologic analysis revealed extenuatedhyperemia, decreased inflammatory cell infiltration and normal alveolar architectureof lung tissue following treatment with baicalin. Analysis of bronchoalveolar lavage(BAL) fluid revealed that treated mice with baicalin led to significantly lessneutrophil influx into the airway, a marked reduction in IL-1β, TNF-α and IL-6concentrations in BAL fluids.In summary, baicalin inhibits the cell lytic activity of Hla via preventing theself-assembling of Hla heptameric pore; baicalin prevents Hla mediated A549cellinjury; baicalin protects mice from S. aurues pneumonia. Our study will lay thefoundation for clarifying the anti-infective mechanism of baicalin and developmentof innovative anti-infective drugs.更多还原