铜绿假单胞菌PA2800和znuA基因功能的研究

发布时间：2018-06-09 22:54

本文选题：铜绿假单胞菌 + PA2800　；参考：《西北大学》2012年硕士论文

【摘要】：铜绿假单胞菌(Pseudomonas aeruginosa)是一种革兰氏阴性条件致病菌,可在人群中引起严重的急性和慢性感染,由于它对很多抗生素都具有内在耐药性,使其临床治疗非常困难,因此研究铜绿假单胞菌的耐药性和致病性机理对于控制其感染具有重要意义。实验室前期通过转座突变的方法构建了包含17000多个克隆的铜绿假单胞菌随机转座突变体库,用实验室常用的七种抗生素对转座突变体库进行了筛选,得到43个与耐药性相关的基因,其中PA2800转座突变体对四环素和环丙沙星的敏感性增加,本论文在此基础上对该基因的功能做了进一步研究。研究发现,突变体PAO(Δ2800)相较于野生型PAO1对Tc的敏感性增加了4倍,对Cip和Cm的敏感性增加了2倍,PA2800互补菌株对抗生素的敏感性与野生型相当,而PA2800过表达菌株对Tc的敏感性降低,但对其他抗生素无变化。结果表明,基因PA2800确实与铜绿假单胞菌对Cip, Cm和Tc三种抗生素的敏感性相关。同时,PA2800过表达会增强铜绿假单胞菌对季铵类化合物的抗性,可提高8倍。 PA2800编码的产物与脂蛋白VacJ有49%的相似性,vacJ在大肠杆菌和志贺氏菌中与其致病性相关。通过果蝇感染实验发现,突变体PAO(Δ2800)对感染果蝇的致死率要高于野生型PAO1。前期研究发现,oprH和znuA在突变体PAO(Δ2800)中的表达升高。oprH是受低镁浓度调节的基因,该基因的高表达会增加对氯霉素和喹诺酮类的吸收,通过实验证明PAO(Δ2800)对Cm和Cip敏感性增加与oprH的高表达相关。已有研究表明znuA基因在大肠杆菌、耶尔森菌和布鲁杆菌等的致病性中有重要作用。在铜绿假单胞菌中构建了基因znuA的突变体PAO(ΔznuA)和双突变体PAO(Δ2800ΔznuA),在果蝇感染实验中,这两种突变体对果蝇的致死率相较于野生型明显降低。由此可见,突变体PAO(A2800)的致病性增强与znuA的高表达有着密切的关系。在铜绿假单胞菌网站中预测ZnuA为可能的粘附因子,通过粘附性实验发现PAO(AznuA)的粘附性明显低于野生型PAO1,表明该基因确实与粘附性相关。znuA的表达受高Zn2+浓度的抑制,低Zn2+浓度对其表达无影响。而低浓度的Mn2+、Cu2+和Fe2+会促进znuA的表达,可提高约两倍,Cd2+对znuA的表达无影响。突变体PAO(ΔznuA)在基础培养基中的生长不受无锌条件的影响。通过外膜稳定性实验来检测PA2800编码的外膜蛋白对外膜的影响,实验发现,PAO(A2800)在含EDTA平板上的生长受到明显的抑制,表明该突变体的外膜稳定性受到破坏,所以PA2800对维持铜绿假单胞菌外膜的稳定性有重要作用。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a gram-negative conditional pathogen that can cause severe acute and chronic infections in people. Because of its inherent resistance to many antibiotics, its clinical treatment is very difficult. Therefore, it is important to study the drug resistance and pathogenicity of Pseudomonas aeruginosa to control its infection. A random transposition mutants library containing more than 17000 clones of Pseudomonas aeruginosa was constructed by transposition mutation in early laboratory. Translocation mutants were screened with seven antibiotics commonly used in laboratory, and 43 genes related to drug resistance were obtained, among which PA2800 translocation mutants were more sensitive to tetracycline and ciprofloxacin. In this paper, the function of the gene was further studied. It was found that the sensitivity of mutant PAO (螖 2800) to Tc was four times higher than that of wild type PAO1. The sensitivity to Cip and cm increased by 2 times, and the sensitivity of PA2800 to antibiotics was similar to that of wild type, but the sensitivity of PA2800 overexpressed strain to Tc was decreased, but not to other antibiotics. The results showed that the gene PA2800 was indeed related to the susceptibility of Pseudomonas aeruginosa to three antibiotics, Cip, cm and Tc. The overexpression of PA2800 increased the resistance of Pseudomonas aeruginosa to quaternary ammonium compounds by 8 times. The product encoded by PA2800 had 49% similarity with lipoprotein VacJ and was associated with pathogenicity in Escherichia coli and Shigella. It was found that the mortality of the mutant PAO (螖 2800) was higher than that of wild type PAO1.Prophase studies showed that the expression of Pao (螖 2800) and Pao (螖 2800) in the mutant PAO (螖 2800) were higher than those of wild type PAO1.The results showed that PAO (螖 2800) was a gene regulated by low mg concentration. The overexpression of this gene increases the absorption of chloramphenicol and quinolones. The results of experiments show that the increased sensitivity of PAO (螖 2800) to cm and Cip is related to the overexpression of OPRH. It has been shown that znuA gene is present in Escherichia coli. Yersinia and Brucella play an important role in pathogenicity. The mutant PAO (螖 ZnuA) and the double mutant PAO (螖 2800 螖 ZnuA) were constructed in Pseudomonas aeruginosa. In the experiment of Drosophila infection, the fatality rate of these two mutants to Drosophila was significantly lower than that of wild type. Therefore, the increased pathogenicity of the mutant PAOOA2800 was closely related to the high expression of znuA. ZnuA was predicted to be a possible adhesion factor in Pseudomonas aeruginosa website. It was found that the adhesion of AznuA was significantly lower than that of wild type PAO1, which indicated that the expression of ZnuA was inhibited by high Zn2 concentration, but it was not affected by low Zn2 concentration. However, low concentrations of mn _ 2, Cu _ 2 and Fe _ 2 promoted the expression of znuA, and increased the expression of znuA by about twice as much as CD _ 2. The growth of the mutant PAO (螖 znuA) in the basal medium was not affected by the zinc-free condition. The effects of the outer membrane protein encoded by PA2800 on the external membrane of PA2800 were detected by the external membrane stability test. It was found that the growth of PAO (螖 znuA) on the plate containing EDTA was significantly inhibited. The results indicated that the outer membrane stability of the mutant was destroyed, so PA2800 played an important role in maintaining the outer membrane stability of Pseudomonas aeruginosa.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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