【作者】 杨雅麟；

【导师】 王建华；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2009， 博士

【摘要】 菌丝霉素(Plectasin)是从腐生子囊菌(the saprophytic ascomycete Pseudoplectania nigrella)中分离得到首例真菌防御素,Plectasin具有有效的抗革兰氏阳性菌且无溶血性等功能,是一种具有治疗潜能的肽抗生素。本文重点研究了Plectasin的大肠杆菌高效融合表达、性质功能、二硫键构效关系及其抑菌机理。取得主要结果如下:1)通过整合三个影响表达的关键策略:抗菌肽基因的宿主密码子优化、硫氧还蛋白融合表达和柱上切割,成功实现了一个富含二硫键Plectasin基因在大肠杆菌中的高效表达。融合蛋白Trx-Plectasin的表达水平为细胞总蛋白的53.6%,约58.5%的融合蛋白以可溶形式表达。经亲和层析纯化到均一的融合蛋白。采用Xa因子柱上酶切融合蛋白有效获得重组的Plectasin,质谱分析重组Plectasin的分子量与其理论分子量相符。经凝胶过滤层析和反相高效液相色谱有效获得高纯度的plectasin。2)从折叠、氧化还原、热处理、离子影响、最小抑菌试验和溶血性试验等方面研究了Plectasin的性质及功能。Plectasin在含有鏊合剂EDTA和甘油的TGE缓冲液中折叠效率远高于在HAc缓冲液中折叠效率,在HAc缓冲液中添加Arg对Plectasin的折叠有一个重要的正效应。氧化还原和热处理结果表明破坏Plectasin二硫键影响其稳定性进而影响其抗菌活性。离子影响实验结果表明一价阳离子对Plectasin的抗菌活性没有影响,二价阳离子能强烈影响Plectasin的抗菌活性,其中钙离子的影响明显比镁离子强;最小抑菌实验结果表明重组的plectasin可以有效抑制革兰氏阳性菌,但对革兰氏阴性菌和丝状真菌基本没有抗菌活性。溶血实验结果表明Plectasin对兔的红细胞无溶血性。3)采用丙氨酸突变成对半胱氨酸的方法设计Plectasin二硫键突变体,结合生物信息学工具对其一级和二级结构参数进行预测,同时采用毕赤酵母X-33分泌重组表达Plectasin及其二硫键突变体,研究不同二硫键对Plectasin抗菌活性的影响,结果表明不同二硫键对Plectasin的抗菌活性影响程度不同, C1-C4二硫键对其抗菌活性影响最大,C3-C6二硫键影响次之,C2-C5影响最小。4)通过离子影响试验、形态学观察以及凝胶阻滞试验,初步推论Plectasin对金黄色葡萄球菌的抑菌机理为:Plectasin不具有与细胞内物质DNA结合的能力,其与细胞表面的作用方式并非简单的静电相互吸引作用,而主要通过特异性与细胞壁上的二价阳离子结合位点如磷壁酸(TA)和脂磷壁酸发生反应,竞争性取代在细胞壁起桥梁和电荷中和作用的二价阳离子,如Ca2+和Mg2+离子,1)可能导致细胞壁紊乱,增加了细胞壁的渗透性,形成孔洞,内容物渗漏,最后导致整个细胞的裂解死亡;2)也可能是通过取代和激活自溶素,导致溶菌酶的失控降解,使细胞自发裂解死亡。总之,本研究成功实现了一个富含二硫键Plectasin基因在大肠杆菌中的高效表达,为生产富含二硫键的防御素提供了一个有效的平台;证实了Plectasin二硫键影响其稳定性进而影响其抗菌活性,且不同二硫键对Plectasin的抗菌活性影响的程度不同;提出了Plectasin特异性结合细胞壁上的二价阳离子结合位点如磷壁酸(TA)和脂磷壁酸的抑菌机理,为阐明Plectasin的作用机制奠定基础。这些结果对于抗革兰氏阳性菌病特别是链球菌病的预防药物新产品的开发提供理论依据,促进Plectasin在防控链球菌病上的应用。更多还原

【Abstract】 Plectasin with potent activity against Gram-positive bacteria but no hemolytic activity, the first defensin-type antimicrobial peptide isolated from a saprophytic fungus, was an inoffensive antibiotic with therapeutic potential.The Escherichia coli (E. coli) fusion expression, characterization, functions, the disulfide structure-activity relationship and anti-Staphylococcus aureus (S. aureus) mechanism of plectasin were studied in this paper. The main results are as follows:1) In this study, we present the high-level expression of Cys-rich plectasin in E. coli by the integration of three key strategies: codon usage bias, fusion partner and on-column cleavage. The expression level of the fusion protein Trx-plectasin accounted for 53.6% of cellular protein, and about 58.5% of the target proteins were in a soluble form. The soluble fusion protein was easily purified to near homogeneity by affinity chromatography using hexahistidine tag. Recombinant plectasin was effectively obtained by on-column cleavage of the fusion protein with factor Xa. The molecular mass of recombinant plectasin determined by MALDI-TOF (matrix assisted laser desorption ionization-time-of-flight) is equal to its theoretical molecular weight. High purity plectasin was achieved by gel filtration chromatography and RP-HPLC.2) The characterization and functions of plectasin was studied from folding, oxidation and reduction, heat treatment, ion-effect tests and minimal inhibitory assays and hemolytic assays. Recombinant plectasin with three cysteine bridges can be properly refolded in TGE buffer or in 0.01% acetic acid with 5 M Arg. Oxidation, reduction and heat treatment of plectasin showed that the destruction of the plectasin disulfide bonds affect the stability of its conformation and therefore lost its antimicrobial activity. Antimicrobial activity assays showed that plectasin was active in vitro against Gram-positive bacteria, but showed no or limited activities against Gram-negative bacteria and fungi. The effects of the different valent cations on the anti-S. aureus activity of plectasin were different. Monovalent cation had no effect on the antimicrobial activity of plectasin. However, divalent cations had a stronger negative effect on the antimicrobial activity of plectasin. These results also indicate that the antimicrobial activity of plectasin was more sensitive to calcium ions than magnesium ions. Hemolysis assays demonstrated that plectasin was not hemolytic for rabbit erythrocytes.3) For test the role of the disulfide array in the antimicrobial activity of plectasin, we have adopted a site-directed mutagenesis approach to generate seven paired Ala for Cys amino acid substitutions corresponding to plectasin with one, two or three disulfide bridges. With bioinformatics tools, the parameters of the primary and secondary structure of mutations that disrupted disulfide bonds were predicted. Plectasin and its mutations were expressed in Pichia pastoris expression system. Paired Ala for Cys amino acid substitutions in plectasin were tested for effects on antimicrobial activity. The results showed that the disulfide bonds have different effects on antimicrobial activity. The greatest effect on the antimicrobial activity of plectasin was the disulfide bond of C1-C4, the next effect was C3-C6, and the least effect was C2-C5. 4) Through the ion-effect test, morphology observation, and gel retardation experiments, the anti-S. aureus mechanism of plectasin was preliminaryly inferenced as follows:Plectasin can not bond to the plasmid DNA. The mode of action with cell wall is not a simple electrostatic attraction, and mainly through the specificity interaction with the divalent cations binding place in the cell wall of S. aureus, such as teichoic acid (TA) and lipoteichoic acid, competitively replace divalent cations, as Ca2 + and Mg2 +, which plays a bridge and charge neutralization role. 1) The plectasin may disrupt the cell wall and increase the permeability of the cell wall, resulting in pore formation and leakage of contents, and eventually led to the death of a whole cell lysis; 2) The plectasin may also be displace and activate autolysins leading to uncontrolled degradation of the muramidase layer and often spontaneous lysis of the cytoplasmic membrane.In short, the successful expression of a disulfide-rich plectasin gene in E. coli in this study would provide an efficient and facile platform for the production or study of disulfide-rich AMPs. This study confirmed that the destruction of the plectasin disulfide bonds affect the stability of its conformation and therefore lost its antimicrobial activity, and the disulfide bonds have different effects on antimicrobial activity. This study also inferenced that the anti-S. aureus mechanism of plectasin was through the specificity interaction with the divalent cations binding place in the cell wall of S. aureus. It help to clarify the antimirobial mechanism of plectasin. These results would provide a theoretical basis for the development of new prevention drug products with anti-Gram-positive bacteria activity, and promote the applications of plectasin on the control of streptococcus disease.更多还原