β-谷甾醇和毛蕊花糖苷对肺炎链球菌溶血素的抑制作用及机制

发布时间：2018-03-21 00:49

  本文选题：肺炎链球菌　切入点：溶血素　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：肺炎链球菌(Streptococcus pneumoniae)是一种革兰氏阳性微生物,可引起多种感染的重要病原体,其在机体上呼吸道定植,并在世界范围内引起一系列致命性感染,如肺炎、中耳炎、菌血症和脑膜炎等。肺炎链球菌也是导致社区获得性肺炎和患者住院治疗的主要病原体之一,在婴幼儿、老年人及其他疾病患者中发病率极高,诱发条件包括如无脾、慢性疾病和免疫缺陷性疾病。传统抗生素被认为是肺炎链球菌感染临床治疗的第一选择,但随着全球抗生素滥用导致抗生素耐药菌的出现,细菌耐药性已经加快了肺炎链球菌的进化和传播,其中β-内酰胺类抗生素的抗性分布尤为广泛,对青霉素不敏感的肺炎链球菌分离株在2004年时已经达到35%,此外,对氯喹诺酮和大环内酯类不敏感的菌株引起的病例数量也在增加。同时,肺炎链球菌作为一种耐药细菌,甚至已经对万古霉素产生耐药性。肺炎链球菌溶血素(pneumolysin,PLY)是肺炎链球菌现已明确的主要毒力因子之一。这一毒素是胆固醇依赖性溶细胞素的成员,胆固醇参与诱导肺炎链球菌溶血素形成多亚基复合物,即多聚体,通过在膜上形成孔而裂解宿主细胞。目前对溶血素的致病性研究已十分清晰,PLY是肺炎链球菌必需的毒力因子,可以穿透宿主细胞的物理防御、刺激宿主细胞凋亡、具有炎症性质,并激活补体系统。除红细胞外,PLY还可作用于机体上皮细胞、巨噬细胞,具有免疫逃避和损伤DNA链的作用。不表达PLY的肺炎链球菌缺失菌株致病力显著降低,在感染试验动物中仅表现为弱毒性,表明溶血素在肺炎链球菌引发的侵袭性感染中有利于增强其致病力。PLY在肺炎球菌致病过程中发挥的重要作用使得该毒力因子成为治疗肺炎球菌感染的最有潜力的靶标之一。药用植物中的天然化合物为药物研发提供了广泛来源,而依据抗毒力策略针对病原体主要毒力因子进行的药物筛选也主要依赖于天然化合物。本研究以肺炎链球菌PLY为研究靶标,从多种药用植物有效成分中筛选出β-谷甾醇和毛蕊花糖苷两个天然化合物单体,通过红细胞裂解试验、最小抑菌浓度测试、免疫印迹分析、寡聚化分析、以蛋白3D结构为基础的计算生物学分析、药物对肺上皮细胞的保护效果以及对肺炎链球菌肺炎小鼠模型的保护作用,对两种受试药物中和PLY生物学毒性的能力和机制进行了分析,为天然化合物抗毒力防控病原体感染,降低抗生素对微生物的生存压力,减缓细菌多药耐药进化进程提供了研究基础。研究中使用纯化的重组蛋白(recombinant PLY,r PLY),在十余个天然化合物中筛选得到的β-谷甾醇和毛蕊花糖苷均可在较低剂量下直接中和r PLY对红细胞的裂解作用。将受试药物与肺炎链球菌野生型菌株D39共培养,药物浓度高于2048μg/ml时依然不影响细菌生长,表明β-谷甾醇和毛蕊花糖苷对肺炎球菌不具备抗菌活性。收集与受试化合物共培养菌液中的菌体进行Western Blot试验,PLY的特异性条带粗细均一,结果表明随着药物浓度增高,细菌表达PLY的能力不受影响,即受试药物不影响肺炎球菌PLY的基因转录和翻译过程,其抑制作用是直接与毒素特定结构结合使PLY失活或封闭活性位点的结果。本研究为进一步探讨此两种化合物抑制PLY溶细胞活性的机制,应用分子对接和分子动力学模拟的方法,以已知3D结构的同源蛋白为模板构建PLY的结构模型,分别与β-谷甾醇和毛蕊花糖苷进行对接,选择优势构象,并对结果实施分子动力学计算。评估数据后结果表明,游离PLY在溶液中活动自由,蛋白与毛蕊花糖苷结合后显示较弱的灵活性,表明PLY残基由于与毛蕊花糖苷结合后增加刚性,毛蕊花糖苷结合在PLY结构域3和结构域4之间的裂口处,该区域在蛋白寡聚过程中发挥作用,结合位点处的稳定性主要是残基Asp471、Asn470、Glu277、Tyr358和Arg359所提供;β谷甾醇-PLY复合物中,由于β-谷甾醇C25的烷基链引起相对较大空间位阻,阻碍了化合物与PLY残基Thr459、Leu460之间的密切相互作用,它和Thr459、Leu460之间的距离长于蛋白与天然受体胆固醇的距离,但作为胆固醇结构类似物β-谷甾醇与PLY的结合方式基本与胆固醇一致。PLY属于胆固醇依赖性细胞毒素,目前对该家族蛋白已有深入研究。较高浓度的PLY可以无需借助细胞膜或胆固醇在溶液中自发组装形成多聚体,我们通过高效液相色谱法,分析预处理过的药物-蛋白混合样品,结果指出β-谷甾醇不影响PLY的自寡聚,而毛蕊花糖苷则显著抑制PLY寡聚体的形成,这也与我们计算生物学的结果一致。肺炎球菌D39引起肺上皮细胞凋亡,而PLY可直接引起肺上皮细胞、巨噬细胞等裂解坏死。PLY具有基因毒性,能够在不严重破坏宿主细胞膜的情况下诱导DNA损伤,造成核内离散的γ-H2AX病灶。本研究将PLY与人肺腺癌上皮细胞A549共培养样品按浓度梯度分别混合β-谷甾醇和毛蕊花糖苷,使用激光共聚焦仪器观察荧光染色后样品中细胞状态、并评估乳酸脱氢酶释放量,实验结果表明胆固醇类似物β-谷甾醇和针对寡聚化结构域的毛蕊花糖苷均可在较低浓度下显著抑制PLY对A549细胞损伤,降低靶细胞坏死率。免疫荧光实验显示,在受试药物存在条件下暴露于PLY的A549细胞核出现DNA损伤的频率显著降低。这些结果表明,通过阻止PLY的寡聚化或阻止其与膜胆固醇的初始结合,可抑制其对真核上皮细胞的细胞毒性、减弱遗传毒性。建立肺炎链球菌肺炎小鼠模型后,考察β-谷甾醇和毛蕊花糖苷对肺炎球菌肺炎的治疗效果。实验结果表明,β-谷甾醇和毛蕊花糖苷的治疗均可显著提高致死性肺炎小鼠的存活率,降低肺炎链球菌感染小鼠肺部细菌定植数,有利于机体对病原体的清除,缓解实验小鼠的肺组织充血、炎症和损伤症状。综上所述,β-谷甾醇结合于PLY胆固醇结合位点阻碍毒素与膜的结合、毛蕊花糖苷结合在PLY寡聚化相关位点抑制其多聚成孔,从而抑制了毒素的细胞毒性和基因毒性、保护靶器官上皮细胞、缓解侵袭性感染对机体的损伤、为机体提供保护,从而在药物无抗菌活性的情况下起到有效的抗感染效果。本研究为药用植物有效成分的药理活性提供了补充,并为依据抗毒力思路抗感染提供了理论基础和先导化合物。
[Abstract]:Streptococcus pneumoniae (Streptococcus pneumoniae) is a gram positive microorganisms, can cause a variety of important pathogen infection of the respiratory tract, the colonization, and caused a series of fatal infections in the world, such as pneumonia, otitis media, bacteremia and meningitis. Streptococcus pneumoniae is one of the leading and the main pathogens of pneumonia and hospitalization patients with community acquired in infants, high incidence of elderly people and patients with other diseases, such as splenic induced conditions, chronic diseases and immunodeficiency disease. Traditional antibiotics is regarded as the first choice for the clinical treatment of Streptococcus pneumoniae infection, but with the global overuse of antibiotics leads to the emergence of antibiotic resistant bacteria, bacterial resistance has accelerated the evolution and spread of Streptococcus pneumoniae, the distribution of resistance of beta lactam antibiotics is particularly widespread, not sensitive to penicillin In addition plant in 2004 has reached 35%, the number of Streptococcus pneumoniae isolates, which is not sensitive to chlorine quinolones and macrolides strains were also increased. At the same time, Streptococcus pneumoniae as a resistant bacteria, even resistant to vancomycin. Pneumonia streptococcus hemolysin (pneumolysin, PLY) is one of the main virulence it is clear that the factor of Streptococcus pneumoniae. This toxin is a cholesterol dependent soluble cytokine members, cholesterol is involved in the induction of pneumolysin formed Doyaki complexes, namely multimers, by forming pores in the membrane and lysis of host cells. The present study on the pathogenicity of hemolysin has been very clear, PLY is essential for Streptococcus pneumoniae virulence factors, physical defense can penetrate the host cells, stimulate the apoptosis of host cells, with inflammatory properties, and activate the complement system. In addition to red blood cells, but also PLY Acting on the body has the function of epithelial cells, macrophages, immune escape and damage of DNA chain. The expression of PLY of Streptococcus pneumoniae pathogenicity mutant strain decreased significantly in animal infection test showed only weak toxicity, that caused hemolysin of Streptococcus pneumoniae infection in favor of an important role to enhance the pathogenicity of.PLY in the play the pathogenesis of pneumococcal virulence factors become one of the most promising target for the treatment of pneumococcal infection. Natural compounds from medicinal plants provides a wide range of sources for drug development, drug screening and the basis of the anti virulence strategy for main virulence factors of pathogens also rely mainly on natural compounds. In this study, PLY of Streptococcus pneumoniae the target selected p-sitosterol and Mao Ruihua glucoside two natural compounds from various effective ingredients in medicinal plants, The red blood cell lysis test, minimum inhibitory concentration test, Western blot analysis, oligomerization analysis, calculation and analysis of biological 3D by protein structure based drug, protective effect on lung epithelial cells and the protective effect of Streptococcus pneumoniae pneumonia in mice model, the ability and mechanism of two kinds of tested drugs and biological toxicity of PLY the analysis for natural compounds and anti virulence of pathogen infection, reduce antibiotic on microbial survival pressure, reduce bacterial multidrug resistance evolution process provides the basis of the research. In the study of purification of recombinant proteins using (recombinant PLY, R PLY), cracking screened at more than ten natural compounds in p-sitosterol and acteoside can at lower doses of neutralization R PLY on red blood cell. The tested drug and Streptococcus pneumoniae in the wild type strain D39 were cultured, the drug concentration was higher than 2 048 g/ml still does not affect the growth of bacteria, showed that beta sitosterol and verbascoside of pneumococcus has no antibacterial activity. The collection of Western Blot test and the test compounds were cultured in liquid cell, PLY specific band of uniform thickness, the results show that with the drug concentration increased, bacterial expression ability of PLY is not affected, the tested drugs did not affect the transcription and translation of pneumococcal PLY, its inhibitory effect is directly combined with the specific structure of the toxin or inactivation of PLY closed active site results. This study further explores the mechanism of the two kinds of compounds that inhibit PLY cytolytic activity, molecular docking and molecular simulation method the dynamics, with known structure of 3D homologous protein structure model template construction of PLY, respectively for docking with the beta sitosterol and verbascoside, choose the advantage and application of fruit conformation. The molecular dynamics calculation. After evaluating the data. The results show that the free PLY activity in solution, binding protein and verbascoside showed weak flexibility, showed that PLY residues due to the combination and verbascoside increased rigidity, verbascoside in combination with PLY domain 3 and domain split between the 4, play the role of the region in the protein oligomerization process, combined with the stability of site is mainly the residues Asp471, Asn470, Glu277, Tyr358 and Arg359; beta sitosterol -PLY complexes, the alkyl chain of p-sitosterol C25 due to relatively large steric hindered compounds and PLY residues Thr459 a close interaction between Leu460, Leu460 and Thr459 it, the distance between the protein and the natural receptor of cholesterol is longer than the distance, but the combination of cholesterol analogues as beta sitosterol and PLY and cholesterol..PLY belongs to Cholesterol dependent cell toxin, the in-depth study of the protein family. High concentrations of PLY will be without the help of cholesterol in the cell membrane or solution self assemble to form multimers, we by the high performance liquid chromatography analysis of drug - treated protein mixed samples, the results pointed out that since the oligomeric beta sitosterol does not affect PLY formation, and verbascoside inhibited PLY oligomers, which is also our computational biology results. Due to pneumococcal D39 apoptosis of lung epithelial cells, and PLY can directly cause lung epithelial cells, macrophages and.PLY lysis necrosis has genetic toxicity, can induce DNA damage without serious damage to the host cell membrane under the condition caused by gamma -H2AX lesions discrete nucleus. In this study, PLY and human lung adenocarcinoma A549 cells co cultured samples were mixed according to the concentration gradient of - sitosterol and Mullein sugar By using laser confocal instrument to observe the cell state after fluorescence staining in samples, and to assess the lactate dehydrogenase release, the experimental results show that cholesterol analogues of - sitosterol and the oligomeric verbascoside domain can be in low concentration significantly inhibited the PLY damage of A549 cells, reduce the necrosis rate of target cells. Immunofluorescence experiments revealed that in the presence of drug exposure to PLY A549 nuclear DNA damage frequency decreased significantly in subjects. These results show that by blocking PLY oligomerization and membrane cholesterol or prevent the initial combination, can inhibit the epithelial cells of the eukaryotic cell toxicity, genetic toxicity. A weakened pneumococcal pneumonia mice model, investigate therapeutic effect of beta sitosterol and verbascoside in pneumococcal pneumonia. The experimental results show that the beta sitosterol and acteoside treatment significantly 鎻愰珮鑷存�绘�ц偤鐐庡皬榧犵殑瀛樻椿鐜，

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