细菌群体感应信号分子合成酶AmbB的结构生物学研究

发布时间：2018-10-15 18:16

【摘要】：随着临床上抗生素的广泛使用,细菌已出现耐药性,因此寻找新的抗感染途径已成为当今研究的热点。群体感应(quorum sensing, QS)作为细菌间传递信号的重要方式,可释放自诱导物分子来调控多种毒力因子的释放以及细菌的致病性。如今,QS已经成为新型、抗耐药菌药物设计的重要靶标,因该药物不影响细菌生长只抑制QS调控的致病行为,理论上不会引起细菌的耐药性。铜绿假单胞菌是一种可引起呼吸道感染、肺炎等疾病的条件致病菌,其存在三种基于信号分子的QS系统：las、rhl和pqs系统。其中,las系统处于核心地位,它可以调控rhl和pqs的基因表达。但是,随着对铜绿假单胞菌QS系统的深入研究发现,las在群体感应调节中的核心地位并不是绝对的。研究显示非核糖体肽合成酶AmbB参与合成一种新型QS信号分子IQS,它在环境压力下(如低磷环境)取代las进而对rhl、pqs系统进行调控。本论文拟通过生物化学的方法,结合X射线晶体学的研究手段对AmbB蛋白的C端结构域进行研究,以期从结构生物学角度阐释群体感应信号分子IQS的产生机制,亦或为今后群体感应抑制剂的研发提供理论基础。本研究结果包括：1.成功构建表达载体,并利用原核系统表达蛋白,已获得大量高纯度AmbBc蛋白；2.筛选到10种AmbBc晶体生长条件,经优化成功得到用于衍射的单晶；3.收集到分辨率高达2.5A的数据；4.成功制备重金属衍生物及硒代甲硫氨酸晶体；5.通过定点突变引入甲硫氨酸增强反常散射信号解决相位问题,目前蛋白结构已成功解析。
[Abstract]:With the widespread use of antibiotics in clinic, bacteria have become resistant to infection, so finding new ways to resist infection has become a hot research topic. As an important way of transmitting signals between bacteria, colony sensing (quorum sensing, QS) can release self-inducer molecules to regulate the release of many virulence factors and the pathogenicity of bacteria. Nowadays, QS has become an important target for the design of new drug resistant bacteria, because the drug does not affect the growth of bacteria, only inhibit the pathogenic behavior of QS regulation, so it will not cause drug resistance of bacteria in theory. Pseudomonas aeruginosa is a kind of conditional pathogen which can cause respiratory tract infection, pneumonia and other diseases. There are three signal molecule based QS systems: las,rhl and pqs. Among them, the las system is at the core, it can regulate the gene expression of rhl and pqs. However, with the further study of the QS system of Pseudomonas aeruginosa, it is found that the core position of las in the regulation of population induction is not absolute. It has been shown that non-ribosomal peptide synthase (AmbB) is involved in the synthesis of a novel QS signaling molecule, IQS, which replaces las under environmental pressure (e.g. low phosphorus environment) and regulates the rhl,pqs system. In this paper, the C-terminal domain of AmbB protein was studied by means of biochemistry and X-ray crystallography, in order to elucidate the mechanism of IQS production from the point of view of structural biology. Or for the future research and development of group induction inhibitors to provide a theoretical basis. The results of this study include: 1. The expression vector was successfully constructed and expressed in prokaryotic system. A large number of high purity AmbBc proteins were obtained. Ten kinds of AmbBc crystal growth conditions were selected and the single crystals used for diffraction were obtained successfully. 3. Data with a resolution of up to 2. 5 A were collected; 4. 4. Preparation of heavy Metal Derivatives and selenomethionine crystals; 5. The phase problem is solved by introducing methionine enhanced anomalous scattering signal by site-directed mutation, and the protein structure has been successfully analyzed.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R3411

【共引文献】