本文選題：肺炎鏈球菌 + 溶血素�。� 參考：《吉林大學》2017年博士論文

【摘要】：肺炎鏈球菌(Streptococcus pneumoniae,S.pneumoniae)是一種常見的革蘭氏陽性機會致病菌,是社區(qū)獲得性肺炎的重要病原菌。在發(fā)展中國家,每年有近百萬兒童死于肺炎性疾病。除了兒童,老年人和免疫功能不全的人群也都處于肺炎鏈球菌感染的危險之中。肺炎鏈球菌感染可以引起鼻竇炎、中耳炎、肺炎、敗血癥和腦膜炎等相關疾病。近年來,各種抗生素的耐藥菌不斷出現(xiàn)和廣泛傳播,使新型機制抗感染藥物的研發(fā)十分緊迫。隨著對病原菌致病過程的深入了解,抗毒力策略研究顯得尤為重要�？苟玖Σ呗允且约毦亩玖σ蜃訛榘袠�,通過干預細菌的致病過程以達到抗感染的目的。天然化合物是新藥研發(fā)的重要資源庫,通過溶血抑制試驗和熒光共振能量轉移試驗(FRET),本研究篩選到了一種可以同時抑制肺炎鏈球菌溶血素(Pneumolysin,PLY)和分選酶A(Sortase A,SrtA)兩種毒力因子生物學活性的天然化合物,即表沒食子兒茶素沒食子酸酯(Epigallocatechin gallate,EGCG)。EGCG是綠茶茶多酚中的主要活性成分,具有抗氧化、抗炎和抗癌等多種生物藥理學活性。藥敏試驗結果表明,EGCG對肺炎鏈球菌D39的最低抑菌濃度大于≥2234μM(1024μg/ml)。成孔毒素PLY是肺炎鏈球菌的重要毒力因子之一,在肺炎鏈球菌致病過程中發(fā)揮著非常重要的作用。PLY單體可以在富含膽固醇的細胞膜上寡聚,形成圓形的跨膜大孔,使細胞內(nèi)容物外溢,從而導致細胞裂解。PLY的細胞毒性即與其溶細胞活性相關,并且直接與肺炎鏈球菌的致病性相關。本研究通過原核表達和純化獲得了PLY重組蛋白。溶血活性抑制試驗和蛋白質免疫印跡試驗結果表明,EGCG不影響肺炎鏈球菌內(nèi)PLY的表達,但是可以直接抑制PLY的溶血活性。在PLY與人肺泡上皮細胞(A549)的共同孵育體系中加入EGCG,可顯著降低PLY介導的細胞損傷。在體外PLY寡聚化形成體系中加入EGCG,可明顯抑制PLY寡聚體的形成。上述結果表明,EGCG可直接作用于PLY本身而抑制其成孔活性。srta是肺炎鏈球菌的另一重要毒力因子,是廣泛存在于革蘭氏陽性菌細胞膜上的一種轉肽酶。通過識別和切割lpxtg序列,srta可以將許多致病相關的表面蛋白錨定于細胞壁的肽聚糖上。srta的活性直接與肺炎鏈球菌的致病性相關。本研究通過原核表達和純化獲得了srtaΔn81和nana重組蛋白;分別免疫balb/c小鼠后,獲得抗srtaΔn81和nana的高效價血清;通過熒光共振能量轉移試驗檢測了egcg對srta活性的影響。結果發(fā)現(xiàn),egcg可劑量依賴性地抑制srta的肽酶活性;在肺炎鏈球菌培養(yǎng)液中加入不同濃度egcg不影響肺炎鏈球菌內(nèi)srta的表達水平,但可劑量依賴性地降低細菌表面nana的含量;egcg與肺炎鏈球菌共培養(yǎng)可顯著降低細菌生物被膜的形成;在肺炎鏈球菌與人喉癌上皮細胞(hep2)共培養(yǎng)體系中加入egcg,可顯著抑制肺炎鏈球菌對hep2細胞的粘附作用。以上結果表明,egcg通過直接與srta相互作用抑制肺炎鏈球菌內(nèi)srta的轉肽酶活性。通過滴鼻法,我們建立了肺炎鏈球菌感染小鼠肺炎模型,并通過皮下注射egcg對感染小鼠進行治療。結果發(fā)現(xiàn),egcg可明顯延長感染小鼠的存活時間,顯著降低感染小鼠肺部的細菌定殖數(shù);整體和組織病理學觀察顯示,與對照組相比,egcg治療組小鼠肺部的病理性損傷明顯減輕;另外,治療組小鼠的肺濕重/干重比值顯著降低,表明egcg可緩解感染小鼠的肺水腫癥狀。體內(nèi)外功能試驗結果表明,egcg可通過與ply和srta相互作用抑制其生物學活性,從而降低肺炎鏈球菌的致病性。最后,本研究應用分子對接和分子動力學模擬等計算生物學方法,分別獲得了egcg與ply和srta的結合模式及結合位點。結果顯示,egcg通過氫鍵和疏水作用結合于ply第3結構域和第4結構域之間的凹槽,并與ply的ser256、glu277、tyr358和arg359形成較強的相互作用;另外,egcg還可以結合于srta的“活性”區(qū),并與srta的thr169、lys171和phe239形成較強的相互作用,從而封閉活性中心。通過定點殘基突變試驗,我們將這些結合位點的氨基酸分別突變?yōu)楸彼?ala),并且檢測了egcg對這些突變子活性的影響。結果顯示,egcg對ply突變子(ply-e277a、ply-y358a和ply-r359a)溶血活性和細胞毒性的抑制作用明顯降低,對srtaΔn81突變子(srtaΔn81-t169a、srtaΔn81-k171a和srtaΔn81-f239a)肽酶活性的抑制作用也明顯降低。以上實驗結果表明,上述氨基酸殘基突變影響了EGCG與PLY和SrtA的相互作用,這也進一步驗證了分子動力學模擬結果的可靠性。綜上所述,本研究為天然化合物抗感染機制研究提供了重要實驗依據(jù),并且為以PLY和SrtA為靶標進行肺炎鏈球菌抗毒力藥物研究奠定了基礎。
[Abstract]:Streptococcus pneumoniae (S.pneumoniae) is a common gram positive opportunistic pathogen and is an important pathogen of community-acquired pneumonia. In developing countries, nearly a million children die of pneumonia every year. In danger. Streptococcus pneumoniae infection can cause sinusitis, otitis media, pneumonia, septicaemia and meningitis. In recent years, the drug resistant bacteria of various antibiotics have appeared and widely spread, making new mechanism of anti infection drug research and development very urgent. With the deep understanding of the pathogenic process of pathogenic bacteria, the study of anti virulence strategy is obvious. The anti virulence strategy is based on the virulence factor of bacteria as the target and interferes with the pathogenic process of bacteria to achieve the purpose of anti infection. Natural compounds are an important resource pool for new drug research and development. Through hemolysis inhibition test and fluorescence resonance energy transfer test (FRET), this study screened a kind of simultaneous inhibition of pneumonia chain ball. The natural compounds of the biological activity of two kinds of virulence factors, Pneumolysin (PLY) and A (Sortase A, SrtA), namely, epigallocatechin gallate (Epigallocatechin gallate, EGCG).EGCG are the main active components in green tea polyphenols, with antioxidant, anti-inflammatory and anticancer activities. Drug sensitive test The results showed that the minimum inhibitory concentration of EGCG for Streptococcus pneumoniae D39 was greater than 2234 u M (1024 g/ml). The pore toxin PLY is one of the important virulence factors of Streptococcus pneumoniae. It plays a very important role in the pathogenesis of Streptococcus pneumoniae, and.PLY monomer can be oligooligosaccharide on the cell membrane rich in bile solid alcohols, forming a circular transmembrane macropore. The cytotoxicity of cell lysis of.PLY was related to its lysological activity and was directly related to the pathogenicity of Streptococcus pneumoniae. The recombinant protein of PLY was obtained by prokaryotic expression and purification. The results of hemolytic activity inhibition test and protein immunoblot test showed that EGCG did not affect the pneumonia chain. The expression of PLY in cocci can directly inhibit the hemolytic activity of PLY. Adding EGCG to the co Incubating System of PLY and human alveolar epithelial cells (A549) can significantly reduce the PLY mediated cell damage. The addition of EGCG to the PLY oligomer formation system in vitro can obviously inhibit the formation of PLY oligomers. The results show that EGCG can directly act on the formation of PLY oligomers. The inhibition of its orifice active.Srta is another important virulence factor of Streptococcus pneumoniae, a trans peptidase that widely exists on the membrane of Gram-positive bacteria. By identifying and cutting lpxtg sequences, srtA can direct the activity of.Srta on the peptidoglycan of many pathogenetic surface proteins anchored to the cell wall directly with the pneumonia chain. The pathogenicity of Staphylococcus was related. SrtA delta N81 and Nana recombinant protein were obtained by prokaryotic expression and purification. After immunization of balb/c mice, the effective valence serum of anti SrtA delta N81 and Nana was obtained. The effect of EGCG on srtA activity was detected by fluorescence resonance energy transfer test. The results showed that EGCG could inhibit srtA peptides in a dose-dependent manner. Enzyme activity; adding different concentrations of EGCG in Streptococcus pneumoniae culture does not affect the expression level of srtA in Streptococcus pneumoniae, but it can reduce the content of Nana on the surface of bacteria in a dose-dependent manner; co culture of EGCG and Streptococcus pneumoniae can significantly reduce the formation of bacterial biofilm; co culture of Streptococcus pneumoniae and human laryngocarcinoma epithelial cells (Hep2) The adhesion of Streptococcus pneumoniae to Hep2 cells was significantly inhibited by adding EGCG in the system. The above results showed that EGCG inhibited the activity of srtA in Streptococcus pneumoniae by direct interaction with srtA. We established pneumonia model of Streptococcus pneumoniae in mice by dripping nose, and treated infected mice by subcutaneous injection of EGCG. The results showed that EGCG could significantly prolong the survival time of the infected mice and significantly reduce the bacterial colonization of the lungs of the infected mice, and the overall and histopathological observation showed that the pathological damage of the lungs in the EGCG treatment group was significantly reduced compared with the control group, and the lung wet weight / dry weight ratio of the mice in the treatment group decreased significantly, indicating that EGCG could be found in the treatment group. In vitro and in vivo functional test results showed that EGCG could inhibit the biological activity of Streptococcus pneumoniae by interacting with ply and srtA, thus reducing the pathogenicity of Streptococcus pneumoniae. Finally, the combination of molecular docking and molecular dynamics simulation was used to obtain the combination of EGCG with ply and srtA respectively. Patterns and binding sites. The results show that EGCG combines the hydrogen bond and hydrophobic interaction in the groove between the ply third domain and the fourth domain, and forms a stronger interaction with ply's ser256, glu277, tyr358 and arg359; in addition, EGCG can also be combined with the "active" region of srtA and forms a stronger phase with thr169, lys171, and minerals of srtA. Interacts, thus blocking the active center. We mutated the amino acids of these binding sites to alanine (ALA) by a fixed site mutation test, and detected the effect of EGCG on the activity of these mutants. The results showed that EGCG has a inhibitory effect on the hemolytic activity and cytotoxicity of ply mutants (ply-e277a, ply-y358a and ply-r359a). The inhibition of SrtA delta N81 mutants (srtA Delta n81-t169a, srtA Delta n81-k171a and srtA Delta n81-f239a) was also significantly reduced. The above experimental results showed that the mutation of the amino acid residues affected the interaction between EGCG and PLY and SrtA, which further verified the reliability of the molecular dynamics simulation results. The research provides an important experimental basis for the study of the anti infection mechanism of natural compounds, and lays a foundation for the research of anti virulent drugs of Streptococcus pneumoniae with PLY and SrtA as the target.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R378.1

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