EGCG抗肺炎链球菌溶血素和分选酶A的作用及机制

发布时间：2018-05-22 07:45

本文选题：肺炎链球菌 + 溶血素　；参考：《吉林大学》2017年博士论文

【摘要】：肺炎链球菌(Streptococcus pneumoniae,S.pneumoniae)是一种常见的革兰氏阳性机会致病菌,是社区获得性肺炎的重要病原菌。在发展中国家,每年有近百万儿童死于肺炎性疾病。除了儿童,老年人和免疫功能不全的人群也都处于肺炎链球菌感染的危险之中。肺炎链球菌感染可以引起鼻窦炎、中耳炎、肺炎、败血症和脑膜炎等相关疾病。近年来,各种抗生素的耐药菌不断出现和广泛传播,使新型机制抗感染药物的研发十分紧迫。随着对病原菌致病过程的深入了解,抗毒力策略研究显得尤为重要。抗毒力策略是以细菌的毒力因子为靶标,通过干预细菌的致病过程以达到抗感染的目的。天然化合物是新药研发的重要资源库,通过溶血抑制试验和荧光共振能量转移试验(FRET),本研究筛选到了一种可以同时抑制肺炎链球菌溶血素(Pneumolysin,PLY)和分选酶A(Sortase A,SrtA)两种毒力因子生物学活性的天然化合物,即表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)。EGCG是绿茶茶多酚中的主要活性成分,具有抗氧化、抗炎和抗癌等多种生物药理学活性。药敏试验结果表明,EGCG对肺炎链球菌D39的最低抑菌浓度大于≥2234μM(1024μg/ml)。成孔毒素PLY是肺炎链球菌的重要毒力因子之一,在肺炎链球菌致病过程中发挥着非常重要的作用。PLY单体可以在富含胆固醇的细胞膜上寡聚,形成圆形的跨膜大孔,使细胞内容物外溢,从而导致细胞裂解。PLY的细胞毒性即与其溶细胞活性相关,并且直接与肺炎链球菌的致病性相关。本研究通过原核表达和纯化获得了PLY重组蛋白。溶血活性抑制试验和蛋白质免疫印迹试验结果表明,EGCG不影响肺炎链球菌内PLY的表达,但是可以直接抑制PLY的溶血活性。在PLY与人肺泡上皮细胞(A549)的共同孵育体系中加入EGCG,可显著降低PLY介导的细胞损伤。在体外PLY寡聚化形成体系中加入EGCG,可明显抑制PLY寡聚体的形成。上述结果表明,EGCG可直接作用于PLY本身而抑制其成孔活性。srta是肺炎链球菌的另一重要毒力因子,是广泛存在于革兰氏阳性菌细胞膜上的一种转肽酶。通过识别和切割lpxtg序列,srta可以将许多致病相关的表面蛋白锚定于细胞壁的肽聚糖上。srta的活性直接与肺炎链球菌的致病性相关。本研究通过原核表达和纯化获得了srtaΔn81和nana重组蛋白;分别免疫balb/c小鼠后,获得抗srtaΔn81和nana的高效价血清;通过荧光共振能量转移试验检测了egcg对srta活性的影响。结果发现,egcg可剂量依赖性地抑制srta的肽酶活性;在肺炎链球菌培养液中加入不同浓度egcg不影响肺炎链球菌内srta的表达水平,但可剂量依赖性地降低细菌表面nana的含量;egcg与肺炎链球菌共培养可显著降低细菌生物被膜的形成;在肺炎链球菌与人喉癌上皮细胞(hep2)共培养体系中加入egcg,可显著抑制肺炎链球菌对hep2细胞的粘附作用。以上结果表明,egcg通过直接与srta相互作用抑制肺炎链球菌内srta的转肽酶活性。通过滴鼻法,我们建立了肺炎链球菌感染小鼠肺炎模型,并通过皮下注射egcg对感染小鼠进行治疗。结果发现,egcg可明显延长感染小鼠的存活时间,显著降低感染小鼠肺部的细菌定殖数;整体和组织病理学观察显示,与对照组相比,egcg治疗组小鼠肺部的病理性损伤明显减轻;另外,治疗组小鼠的肺湿重/干重比值显著降低,表明egcg可缓解感染小鼠的肺水肿症状。体内外功能试验结果表明,egcg可通过与ply和srta相互作用抑制其生物学活性,从而降低肺炎链球菌的致病性。最后,本研究应用分子对接和分子动力学模拟等计算生物学方法,分别获得了egcg与ply和srta的结合模式及结合位点。结果显示,egcg通过氢键和疏水作用结合于ply第3结构域和第4结构域之间的凹槽,并与ply的ser256、glu277、tyr358和arg359形成较强的相互作用;另外,egcg还可以结合于srta的“活性”区,并与srta的thr169、lys171和phe239形成较强的相互作用,从而封闭活性中心。通过定点残基突变试验,我们将这些结合位点的氨基酸分别突变为丙氨酸(ala),并且检测了egcg对这些突变子活性的影响。结果显示,egcg对ply突变子(ply-e277a、ply-y358a和ply-r359a)溶血活性和细胞毒性的抑制作用明显降低,对srtaΔn81突变子(srtaΔn81-t169a、srtaΔn81-k171a和srtaΔn81-f239a)肽酶活性的抑制作用也明显降低。以上实验结果表明,上述氨基酸残基突变影响了EGCG与PLY和SrtA的相互作用,这也进一步验证了分子动力学模拟结果的可靠性。综上所述,本研究为天然化合物抗感染机制研究提供了重要实验依据,并且为以PLY和SrtA为靶标进行肺炎链球菌抗毒力药物研究奠定了基础。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R378.1

【相似文献】