群体感应系统调控蛋白RsaL的结构及其调节机制的研究

发布时间：2018-05-16 23:21

本文选题：铜绿假单胞菌 + 群体感应系统　；参考：《西北大学》2017年硕士论文

【摘要】：铜绿假单胞菌(Pseudomonas aeruginosa,PA)是一种普遍存在的革兰氏阴性杆菌。它对许多抗生素具有很高的内在抗药性,是病人在医院期间发生感染的第三大致病菌,且很难治愈。因此,寻找新的药物作用靶点开发新型抗致病力药物是目前研究的主要内容。群体感应(Quorum Sensing,QS)是细菌之间交流的一种方式,铜绿假单胞菌中的群体感应系统主要由Las,Rhl和PQS三个系统组成,它们控制着细菌中许多重要的功能。RsaL作为QS的抑制子,与LasR共同调控着QS中信号分子的内态平衡,同时通过调节多种基因的转录水平,影响着毒力、生物被膜(biofilm)等许多重要的表型。然而,其具体的功能及其调节途径并没有被完全阐述。本实验利用ChIP-seq在PAO1全基因组中找到了 RsaL直接结合的两个位点,它们分别位于PA2228/PA2229和pqsH/cdpR的基因间隔区。以前的研究表明,PqsH和CdpR都与PQS信号分子的合成有关。实验结果显示,在rsaL基因的敲除突变体(△rsaL)中pqsH和cdpR基因的表达水平较野生型菌株PAO1下降了很多,从而也降低了 PQS信号分子的产量。rsaL基因的突变导致了 PAO1菌体中生物被膜的降低和绿脓菌素的升高,这些表型主要是由于pqsH或cdpR转录水平的变化引起的。此外,我们解析了 RsaL-DNA复合物的晶体结构,发现RsaL的DNA结合结构域类似于HTH结构,这一结构在许多转录调节子中都是十分保守的。通过定点突变和回补实验,我们确定了 RsaL与DNA结合时起关键作用的氨基酸,它们分别是Arg20,Gln27,Gln38,Gly35,Ser37 和 Ser42。本研究让我们更清楚的理解了 RsaL复杂的调控网络,同时也为探索QS中新的调节蛋白提供了参考和依据。
[Abstract]:Pseudomonas aeruginosa (PA) is a common gram negative bacilli. It has high intrinsic resistance to many antibiotics. It is the third general pathogen infected by the patient in hospital. It is difficult to cure. Therefore, it is currently studied to find new drug targets to develop new anti pathogenic drugs. Quorum Sensing (QS) is a way of communication between bacteria. The quorum induction system in Pseudomonas aeruginosa is composed mainly of three systems, Las, Rhl and PQS. They control many important functional.RsaL in bacteria as the inhibitor of QS, and regulate the internal equilibrium of the signal molecules in QS with LasR, and simultaneously control the internal equilibrium of the signal molecules in QS. By regulating the transcriptional level of multiple genes, it affects many important phenotypes such as virulence, biofilm (biofilm) and many other important phenotypes. However, its specific functions and its regulatory pathways have not been fully explained. This experiment uses ChIP-seq to find two direct binding sites of RsaL in the whole PAO1 genome, which are located in PA2228/PA2229 and pqsH/cdp, respectively. R gene spacers. Previous studies showed that both PqsH and CdpR were related to the synthesis of PQS signal molecules. Experimental results showed that the expression level of pqsH and cdpR genes in the rsaL gene knockout mutant (delta rsaL) decreased a lot more than that of the wild type strain, thus reducing the mutation of the.RsaL gene of the PQS signal molecule resulting in the PAO1. The decrease of biofilm and the increase of Pseudomonas aeruginosa in the bacteria are caused mainly by changes in the pqsH or cdpR transcriptional levels. In addition, we have analyzed the crystal structure of the RsaL-DNA complex and found that the DNA binding domain of RsaL is similar to the HTH structure. This structure is very conservative in the many transcriptional regulators. We have identified the key amino acids in the combination of RsaL and DNA, which are Arg20, Gln27, Gln38, Gly35, Ser37 and Ser42., which let us understand the complex regulatory network of RsaL more clearly, and provide a reference and basis for exploring the new regulator of QS in QS.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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