本文選題：肺炎鏈球菌　切入點：溶血素　出處：《吉林大學》2017年博士論文　論文類型：學位論文

【摘要】：肺炎鏈球菌(Streptococcus pneumoniae)是一種革蘭氏陽性微生物,可引起多種感染的重要病原體,其在機體上呼吸道定植,并在世界范圍內(nèi)引起一系列致命性感染,如肺炎、中耳炎、菌血癥和腦膜炎等。肺炎鏈球菌也是導致社區(qū)獲得性肺炎和患者住院治療的主要病原體之一,在嬰幼兒、老年人及其他疾病患者中發(fā)病率極高,誘發(fā)條件包括如無脾、慢性疾病和免疫缺陷性疾病。傳統(tǒng)抗生素被認為是肺炎鏈球菌感染臨床治療的第一選擇,但隨著全球抗生素濫用導致抗生素耐藥菌的出現(xiàn),細菌耐藥性已經(jīng)加快了肺炎鏈球菌的進化和傳播,其中β-內(nèi)酰胺類抗生素的抗性分布尤為廣泛,對青霉素不敏感的肺炎鏈球菌分離株在2004年時已經(jīng)達到35%,此外,對氯喹諾酮和大環(huán)內(nèi)酯類不敏感的菌株引起的病例數(shù)量也在增加。同時,肺炎鏈球菌作為一種耐藥細菌,甚至已經(jīng)對萬古霉素產(chǎn)生耐藥性。肺炎鏈球菌溶血素(pneumolysin,PLY)是肺炎鏈球菌現(xiàn)已明確的主要毒力因子之一。這一毒素是膽固醇依賴性溶細胞素的成員,膽固醇參與誘導肺炎鏈球菌溶血素形成多亞基復合物,即多聚體,通過在膜上形成孔而裂解宿主細胞。目前對溶血素的致病性研究已十分清晰,PLY是肺炎鏈球菌必需的毒力因子,可以穿透宿主細胞的物理防御、刺激宿主細胞凋亡、具有炎癥性質(zhì),并激活補體系統(tǒng)。除紅細胞外,PLY還可作用于機體上皮細胞、巨噬細胞,具有免疫逃避和損傷DNA鏈的作用。不表達PLY的肺炎鏈球菌缺失菌株致病力顯著降低,在感染試驗動物中僅表現(xiàn)為弱毒性,表明溶血素在肺炎鏈球菌引發(fā)的侵襲性感染中有利于增強其致病力。PLY在肺炎球菌致病過程中發(fā)揮的重要作用使得該毒力因子成為治療肺炎球菌感染的最有潛力的靶標之一。藥用植物中的天然化合物為藥物研發(fā)提供了廣泛來源,而依據(jù)抗毒力策略針對病原體主要毒力因子進行的藥物篩選也主要依賴于天然化合物。本研究以肺炎鏈球菌PLY為研究靶標,從多種藥用植物有效成分中篩選出β-谷甾醇和毛蕊花糖苷兩個天然化合物單體,通過紅細胞裂解試驗、最小抑菌濃度測試、免疫印跡分析、寡聚化分析、以蛋白3D結構為基礎的計算生物學分析、藥物對肺上皮細胞的保護效果以及對肺炎鏈球菌肺炎小鼠模型的保護作用,對兩種受試藥物中和PLY生物學毒性的能力和機制進行了分析,為天然化合物抗毒力防控病原體感染,降低抗生素對微生物的生存壓力,減緩細菌多藥耐藥進化進程提供了研究基礎。研究中使用純化的重組蛋白(recombinant PLY,r PLY),在十余個天然化合物中篩選得到的β-谷甾醇和毛蕊花糖苷均可在較低劑量下直接中和r PLY對紅細胞的裂解作用。將受試藥物與肺炎鏈球菌野生型菌株D39共培養(yǎng),藥物濃度高于2048μg/ml時依然不影響細菌生長,表明β-谷甾醇和毛蕊花糖苷對肺炎球菌不具備抗菌活性。收集與受試化合物共培養(yǎng)菌液中的菌體進行Western Blot試驗,PLY的特異性條帶粗細均一,結果表明隨著藥物濃度增高,細菌表達PLY的能力不受影響,即受試藥物不影響肺炎球菌PLY的基因轉(zhuǎn)錄和翻譯過程,其抑制作用是直接與毒素特定結構結合使PLY失活或封閉活性位點的結果。本研究為進一步探討此兩種化合物抑制PLY溶細胞活性的機制,應用分子對接和分子動力學模擬的方法,以已知3D結構的同源蛋白為模板構建PLY的結構模型,分別與β-谷甾醇和毛蕊花糖苷進行對接,選擇優(yōu)勢構象,并對結果實施分子動力學計算。評估數(shù)據(jù)后結果表明,游離PLY在溶液中活動自由,蛋白與毛蕊花糖苷結合后顯示較弱的靈活性,表明PLY殘基由于與毛蕊花糖苷結合后增加剛性,毛蕊花糖苷結合在PLY結構域3和結構域4之間的裂口處,該區(qū)域在蛋白寡聚過程中發(fā)揮作用,結合位點處的穩(wěn)定性主要是殘基Asp471、Asn470、Glu277、Tyr358和Arg359所提供;β谷甾醇-PLY復合物中,由于β-谷甾醇C25的烷基鏈引起相對較大空間位阻,阻礙了化合物與PLY殘基Thr459、Leu460之間的密切相互作用,它和Thr459、Leu460之間的距離長于蛋白與天然受體膽固醇的距離,但作為膽固醇結構類似物β-谷甾醇與PLY的結合方式基本與膽固醇一致。PLY屬于膽固醇依賴性細胞毒素,目前對該家族蛋白已有深入研究。較高濃度的PLY可以無需借助細胞膜或膽固醇在溶液中自發(fā)組裝形成多聚體,我們通過高效液相色譜法,分析預處理過的藥物-蛋白混合樣品,結果指出β-谷甾醇不影響PLY的自寡聚,而毛蕊花糖苷則顯著抑制PLY寡聚體的形成,這也與我們計算生物學的結果一致。肺炎球菌D39引起肺上皮細胞凋亡,而PLY可直接引起肺上皮細胞、巨噬細胞等裂解壞死。PLY具有基因毒性,能夠在不嚴重破壞宿主細胞膜的情況下誘導DNA損傷,造成核內(nèi)離散的γ-H2AX病灶。本研究將PLY與人肺腺癌上皮細胞A549共培養(yǎng)樣品按濃度梯度分別混合β-谷甾醇和毛蕊花糖苷,使用激光共聚焦儀器觀察熒光染色后樣品中細胞狀態(tài)、并評估乳酸脫氫酶釋放量,實驗結果表明膽固醇類似物β-谷甾醇和針對寡聚化結構域的毛蕊花糖苷均可在較低濃度下顯著抑制PLY對A549細胞損傷,降低靶細胞壞死率。免疫熒光實驗顯示,在受試藥物存在條件下暴露于PLY的A549細胞核出現(xiàn)DNA損傷的頻率顯著降低。這些結果表明,通過阻止PLY的寡聚化或阻止其與膜膽固醇的初始結合,可抑制其對真核上皮細胞的細胞毒性、減弱遺傳毒性。建立肺炎鏈球菌肺炎小鼠模型后,考察β-谷甾醇和毛蕊花糖苷對肺炎球菌肺炎的治療效果。實驗結果表明,β-谷甾醇和毛蕊花糖苷的治療均可顯著提高致死性肺炎小鼠的存活率,降低肺炎鏈球菌感染小鼠肺部細菌定植數(shù),有利于機體對病原體的清除,緩解實驗小鼠的肺組織充血、炎癥和損傷癥狀。綜上所述,β-谷甾醇結合于PLY膽固醇結合位點阻礙毒素與膜的結合、毛蕊花糖苷結合在PLY寡聚化相關位點抑制其多聚成孔,從而抑制了毒素的細胞毒性和基因毒性、保護靶器官上皮細胞、緩解侵襲性感染對機體的損傷、為機體提供保護,從而在藥物無抗菌活性的情況下起到有效的抗感染效果。本研究為藥用植物有效成分的藥理活性提供了補充,并為依據(jù)抗毒力思路抗感染提供了理論基礎和先導化合物。
[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鎬ц偤鐐庡皬榧犵殑瀛樻椿鐜，

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