铜绿假单胞菌CzcCBA主动外排泵的基因调节及PA4489的功能研究

发布时间：2018-06-28 06:20

本文选题：铜绿假单胞菌 + 转座突变　；参考：《西北大学》2007年硕士论文

【摘要】： 铜绿假单胞菌(Pseudomonas aerugionsa)是一种机会致病菌，它可以在人群中引起严重的急性和慢性感染，是病人在医院期间发生感染的第三大致病菌。虽然某些抗生素对铜绿假单胞菌具有一定的治疗作用，但同时，铜绿假单胞菌有一个重要特性是它对许多抗生素具有很高的内在抗药性，这使其成为临床上难以治疗的病原菌。研究铜绿假单胞菌抗药性产生的机理、研究其与致病性相关的基因，对于了解其致病机理和控制其感染极为重要。外排泵对抗生素的外排作用是细菌抗药性的主要原因之一。铜绿假单胞菌中有12个已知和假定的外排泵基因簇。czcCBA基因簇编码其中一个RND外排泵。本实验对这一RND泵的基因调节进行了研究，在重金属盐ZnCl_2存在情况下，用随机突变的方法筛选了基因组中czcCBA基因的调节子。经过测试筛选大约20000个克隆，得到了7个对czcCBA基因表达有影响的突变体。这些突变体与野生型相比，czcCBA表达量发生了很大的变化，在有250nM Zn~(2+)存在的条件下，一个突变体中的表达量比原来提高了5倍，另外6个突变体几乎没有发光，即表达消失。经重组敲除实验及互补实验验证，这7个基因与czcCBA的表达调节有关。先前有研究证明致病菌能够捕获真核宿主细胞以及其他细菌的基因，并整合为自身的基因，从而提高自身对宿主免疫系统的抗性，进而提高致病性。蛋白酶抑制因子α_2巨球蛋白，能够捕获致病菌成功侵袭所需的攻击性蛋白酶，从而作为多细胞动物抵御致病菌的主要防御机制之一。通过比较多细胞动物和许多致病菌的基因序列，发现许多侵袭力更强，能使更高级真核细胞(动物和植物)致病的病原菌具有α_2巨球蛋白的基因(Genome Biology 2004，5：R38)。对基因组的分析发现，在铜绿假单胞菌中也有α_2M基因的同源序列(即PA4489)。但国际上对铜绿假单胞菌中的这个基因的研究几乎没有。本研究的另一个内容就是利用基因置换的方法对铜绿假单胞菌中这一可能的α_2M基因进行了敲除。通过比较野生型和突变株对不同抗生素的抗性差异，发现突变体对多粘菌素的抗性降低，表明yfa-α_2M基因与铜绿假单胞菌对多粘菌素的抗性有关。
[Abstract]:Pseudomonas aerugionsa is a opportunistic pathogen, which can cause severe acute and chronic infection in the population. Although some antibiotics have some therapeutic effects on Pseudomonas aeruginosa, an important characteristic of Pseudomonas aeruginosa is that it has high intrinsic resistance to many antibiotics, which makes it difficult to be treated clinically. It is very important to study the mechanism of drug resistance of Pseudomonas aeruginosa and its pathogenicity related genes in order to understand the pathogenicity mechanism and control the infection of Pseudomonas aeruginosa. The efflux of efflux pump to antibiotics is one of the main reasons of bacterial drug resistance. In Pseudomonas aeruginosa, there are 12 known and assumed output-pump gene clusters. CzcCBA gene cluster encodes one of the RND efflux pumps. In this experiment, the gene regulation of RND pump was studied. In the presence of heavy metal salt ZnCl2, the regulator of czcCBA gene was screened by random mutation method. After testing and screening about 20 000 clones, 7 mutants with influence on czcCBA gene expression were obtained. The expression of czcCBA in one mutant was five times higher than that in the wild type, and the expression of the other 6 mutants was almost no luminescence, that is, the expression disappeared in the presence of 250 nm Zn ~ (2). The results of recombinant knockout and complementary experiments showed that these seven genes were related to the regulation of czcCBA expression. Previous studies have shown that pathogens can capture and integrate the genes of eukaryotic host cells and other bacteria into their own genes, thus increasing their resistance to the host immune system and thus enhancing their pathogenicity. The protease inhibitor 伪 2 macroglobulin can capture the aggressive protease required for the successful invasion of pathogenic bacteria, which is one of the main defense mechanisms of multicellular animals against pathogenic bacteria. By comparing the gene sequences of multicellular animals and many pathogenic bacteria, it was found that many pathogenic bacteria with stronger invasiveness could cause more advanced eukaryotic cells (animals and plants) with 伪 2 macroglobulin gene (Genome Biology 2004 5: R38). The genome analysis showed that the homologous sequence of 伪 -lipo2m gene (PA4489) was also found in Pseudomonas aeruginosa. But there is little international research on this gene in Pseudomonas aeruginosa. Another aspect of this study was to knockout this possible 伪 -S 2M gene in Pseudomonas aeruginosa by gene replacement. By comparing the resistance of wild type and mutant to different antibiotics, it was found that the resistance of mutants to polymyxin decreased, which indicated that yfa- 伪 _ 2 M gene was related to the resistance of Pseudomonas aeruginosa to polymyxin.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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