谷胱甘肽与铜绿假单胞菌致病性及抗生素抗性的关系研究
发布时间：2019-05-19 08:26
【摘要】： 铜绿假单胞菌(Pseudomonas aeruginosa)是病人在医院发生感染的第三大条件致病菌,对宿主细胞引起的氧化伤害,是其导致病人感染死亡的原因之一。谷胱甘肽(glutathione,GSH)是细胞内最重要的抗氧化剂之一,其含量变化与疾病的发生有着密切的关系。资料显示许多肺部疾病如囊肿性纤维化(cysticfibrosis,CF)病人的呼吸道上皮分泌液(epithelial lining fluid,ELF)中,GSH的水平异常低下。由铜绿假单胞菌感染引起的氧化伤害也是导致CF病人症候出现的主要原因之一。因此,在临床实验中已有用吸入GSH的方法来提高CF病人ELF中的GSH水平。另据资料显示,GSH一方面可以减轻抗生素作用细菌时对宿主细胞造成的氧化损害,一方面又消除了一些抗生素所引起的细菌细胞内氧化压力,保护细菌抵御抗生素的抑杀,降低细菌对一些抗生素的敏感性。为了了解GSH在细胞中这种功能上的矛盾以及和铜绿假单胞菌之间的关系,我们深入研究了GSH和铜绿假单胞菌致病性的关系以及对不同抗生素敏感性的影响。 为了研究GSH与铜绿假单胞菌致病性的关系,以荧光素酶基因操纵子luxCDABE发光报道基因构建的致病性因子启动子库为平台,外加不同浓度GSH或化学试剂耗竭细胞内GSH,对启动子库进行筛选比较,依据luxCDABE报道子发光值的变化情况,寻找与GSH有关的致病性因子;构建铜绿假单胞菌谷胱甘肽合成酶基因突变体PAO1(△gshB)和谷胱甘肽还原酶突变体PAO1(△gor),在分子水平进一步确认GSH和这些致病性因子的关系,分析GSH对铜绿假单胞菌致病性的影响。结果发现,GSH对启动子库中exoS、exoY、phzA1和gacA等致病性因子的表达有促进作用,对其余测试过的致病性因子则没有明显影响;铜绿假单胞菌分泌的重要致病因子绿脓菌素(pyocyanin,PCN)的含量,在PAO1(△gshB)中远低于PAO1(P＜0.01);加入GSH后,PCN平均含量在PAO1(△gshB)中从0.47μg/mL提高到1.73μg/mL,差异十分显著(P＜0.01),在PAO1中从1.3μg/mL提高到1.9μg/mL,差异显著(P＜0.05),表明GSH对铜绿假单胞菌生产PCN有促进作用。由于exoS、exoY、phzA1、gacA和PCN与群体感应系统(Quorum-sensing,QS)有着密切的关系,我们对GSH和QS系统的关系进行了相关研究,结果发现,QS系统中rhl系统对gshB和gor基因有负调节作用。除了对以上致病性因子产生影响外,发现铜绿假单胞菌外排泵MexXY-OprM的表达在PAO1(△gshB)中显著降低,并通过互补实验证实是由gshB基因的突变引起的,说明GSH对MexXY-OprM的表达有促进作用。 为了研究GSH对抗生素抗菌活性的影响,利用琼脂扩散方法和最低抑菌浓度(MIC)方法,研究了GSH的存在引起的铜绿假单胞菌对不同抗生素敏感性的变化。结果发现,在LB液体中添加3μg/μL的GSH,铜绿假单胞菌对卡那霉素、红霉素、头孢噻肟钠、环丙沙星、羧苄青霉素和硫酸链霉素的敏感性降低,而对四环素的敏感性增加。铜绿假单胞菌对卡那霉素的MIC由240μg/mL增加为1600μg/mL,对四环素的MIC由25μg/mL降为1.25μg/mL,但是对氯霉素和氨苄青霉素并没有明显地影响。GSSG和GSH具有相同的功能,也可以改变PAO1对抗生素的敏感性。在PAO1(△gor)和PAO1(△gshB)两个突变株中,GSSG和GSH同时可以影响抗生素的敏感性,其程度与野生型相同。上述结果显示,GSH影响铜绿假单胞菌对不同抗生素的敏感性与抗生素的种类密切相关,而与氧化型还是还原型谷光甘肽没有关系,因此其作用可能不是通过改变细胞内的氧化还原状态而起作用的。 总之,GSH一方面对exoS、exoY、phzA1和gacA等致病性基因的表达和PCN的产生起到促进作用,一方面可以改变铜绿假单胞菌对不同抗生素的敏感性。本研究的结果对于GSH生物功能的重新认识、GSH在铜绿假单胞菌感染过程中所扮演的角色,以及铜绿假单胞菌的致病性机理研究等方面具有较大的理论价值和实际意义。
[Abstract]:Pseudomonas aeruginosa is the third-condition pathogen of the patient's infection in the hospital, and the oxidative damage caused by the host cell is one of the causes of the patient's death. Glutathione (GSH) is one of the most important antioxidants in the cell. The results show that the level of GSH is low in many cases of pulmonary diseases, such as cystic fibrosis (CF). The oxidative damage caused by the infection of Pseudomonas aeruginosa is also one of the main causes of the symptoms of CF patients. Thus, in clinical trials, the GSH level in the CF patient ELF has been improved by the method of inhalation of GSH. According to the data, the GSH can reduce the oxidative damage to the host cells when the antibiotics are applied to the bacteria, and on the one hand, the internal oxidation pressure of the bacteria cells caused by the antibiotics is eliminated, the bacteria are protected against the inhibition of the antibiotics, and the sensitivity of the bacteria to some antibiotics is reduced. In order to understand the contradiction between the function of GSH in the cells and the relationship between Pseudomonas aeruginosa, we have studied the relationship between the pathogenicity of GSH and Pseudomonas aeruginosa and the effect on the sensitivity of different antibiotics. In order to study the relationship between the pathogenicity of the GSH and the Pseudomonas aeruginosa, the pathogenic factor promoter library constructed with the luxCDABE luminescent reporter gene of the luciferase gene operon is the platform, and the GSH in the cell is exhausted by the GSH or the chemical agent with different concentration, and the promoter library is screened. According to the variation of the sub-luminescence value reported by luxCDABE, the pathogenic factor related to GSH was found, and the gene mutation of the glutathione synthetase gene of the P. aeruginosa and the glutathione reductase mutant PAO1 (Ogg) were constructed. or), the relationship between GSH and the pathogenic factors is further confirmed at the molecular level, and the pathogenicity of the GSH to the pseudomonas aeruginosa is analyzed, The results showed that GSH could promote the expression of exoS, exoY, phzA1 and gacA in the promoter, and there was no significant effect on the other tested pathogenic factors, and the important pathogenic factors secreted by P. aeruginosa (pylocyanin, PCN) The content of Pol was significantly lower than that of PAO1 (P <0.01), and the average content of PCN increased from 0.47. m/ mL to 1.73. m/ mL in PAO1 (pgshB), and the difference was very significant (P <0.01), and the difference was significant (P <0.01) in PAO1 from 1.3. m u.g/ mL to 1.9. m 05). It is indicated that the production of PCN by the GSH is promoted by the P. aeruginosa. The results show that the rhl system in the QS system has a negative effect on the gshB and gor genes. In addition to the effects on the above pathogenic factors, the expression of MexXY-OprM of the efflux pump of P. aeruginosa was significantly reduced in PAO1 (pgshB) and confirmed by the mutation of the gshB gene, indicating that the expression of GSH on the MexXY-OprM was promoted. In order to study the effect of GSH on the antibacterial activity of antibiotics, the method of agar diffusion and the minimum inhibitory concentration (MIC) were used to study the effects of GSH on the different anti-bacterial activity of P. aeruginosa. The results showed that the sensitivity of GSH and P. aeruginosa to kanamycin, erythromycin, ceftriaxone, ciprofloxacin, penicillins and streptomycin was reduced in LB liquid, and the sensitivity of penicillin and streptomycin to the four rings was reduced. The sensitivity of P. aeruginosa to kanamycin was increased from 240. m u.g/ mL to 1600. m u.g/ mL, and the MIC for tetracycline was reduced to 1.25. m u.g/ mL from 25. m There is no obvious effect. GSSG and GSH have the same function, and PAO1 can also be changed. The susceptibility of antibiotics to antibiotics in two mutants of PAO1 and PAO1, GSSG and GSH can also influence the sensitivity of antibiotics, The results showed that the effects of GSH on the susceptibility of Pseudomonas aeruginosa to different antibiotics were closely related to the species of antibiotics, but not in relation to the oxidized or reduced-type cereal, and therefore the effect might not be to change the redox in the cells. In conclusion, GSH has a catalytic effect on the expression of pathogenic genes such as exoS, exoY, phzA1 and gacA and the production of PCN. On the one hand, the Pseudomonas aeruginosa can be changed. The results of this study have a better understanding of the function of GSH, the role of GSH in the process of Pseudomonas aeruginosa infection, and the pathogenic mechanism of P. aeruginosa.
【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378
[Abstract]:Pseudomonas aeruginosa is the third-condition pathogen of the patient's infection in the hospital, and the oxidative damage caused by the host cell is one of the causes of the patient's death. Glutathione (GSH) is one of the most important antioxidants in the cell. The results show that the level of GSH is low in many cases of pulmonary diseases, such as cystic fibrosis (CF). The oxidative damage caused by the infection of Pseudomonas aeruginosa is also one of the main causes of the symptoms of CF patients. Thus, in clinical trials, the GSH level in the CF patient ELF has been improved by the method of inhalation of GSH. According to the data, the GSH can reduce the oxidative damage to the host cells when the antibiotics are applied to the bacteria, and on the one hand, the internal oxidation pressure of the bacteria cells caused by the antibiotics is eliminated, the bacteria are protected against the inhibition of the antibiotics, and the sensitivity of the bacteria to some antibiotics is reduced. In order to understand the contradiction between the function of GSH in the cells and the relationship between Pseudomonas aeruginosa, we have studied the relationship between the pathogenicity of GSH and Pseudomonas aeruginosa and the effect on the sensitivity of different antibiotics. In order to study the relationship between the pathogenicity of the GSH and the Pseudomonas aeruginosa, the pathogenic factor promoter library constructed with the luxCDABE luminescent reporter gene of the luciferase gene operon is the platform, and the GSH in the cell is exhausted by the GSH or the chemical agent with different concentration, and the promoter library is screened. According to the variation of the sub-luminescence value reported by luxCDABE, the pathogenic factor related to GSH was found, and the gene mutation of the glutathione synthetase gene of the P. aeruginosa and the glutathione reductase mutant PAO1 (Ogg) were constructed. or), the relationship between GSH and the pathogenic factors is further confirmed at the molecular level, and the pathogenicity of the GSH to the pseudomonas aeruginosa is analyzed, The results showed that GSH could promote the expression of exoS, exoY, phzA1 and gacA in the promoter, and there was no significant effect on the other tested pathogenic factors, and the important pathogenic factors secreted by P. aeruginosa (pylocyanin, PCN) The content of Pol was significantly lower than that of PAO1 (P <0.01), and the average content of PCN increased from 0.47. m/ mL to 1.73. m/ mL in PAO1 (pgshB), and the difference was very significant (P <0.01), and the difference was significant (P <0.01) in PAO1 from 1.3. m u.g/ mL to 1.9. m 05). It is indicated that the production of PCN by the GSH is promoted by the P. aeruginosa. The results show that the rhl system in the QS system has a negative effect on the gshB and gor genes. In addition to the effects on the above pathogenic factors, the expression of MexXY-OprM of the efflux pump of P. aeruginosa was significantly reduced in PAO1 (pgshB) and confirmed by the mutation of the gshB gene, indicating that the expression of GSH on the MexXY-OprM was promoted. In order to study the effect of GSH on the antibacterial activity of antibiotics, the method of agar diffusion and the minimum inhibitory concentration (MIC) were used to study the effects of GSH on the different anti-bacterial activity of P. aeruginosa. The results showed that the sensitivity of GSH and P. aeruginosa to kanamycin, erythromycin, ceftriaxone, ciprofloxacin, penicillins and streptomycin was reduced in LB liquid, and the sensitivity of penicillin and streptomycin to the four rings was reduced. The sensitivity of P. aeruginosa to kanamycin was increased from 240. m u.g/ mL to 1600. m u.g/ mL, and the MIC for tetracycline was reduced to 1.25. m u.g/ mL from 25. m There is no obvious effect. GSSG and GSH have the same function, and PAO1 can also be changed. The susceptibility of antibiotics to antibiotics in two mutants of PAO1 and PAO1, GSSG and GSH can also influence the sensitivity of antibiotics, The results showed that the effects of GSH on the susceptibility of Pseudomonas aeruginosa to different antibiotics were closely related to the species of antibiotics, but not in relation to the oxidized or reduced-type cereal, and therefore the effect might not be to change the redox in the cells. In conclusion, GSH has a catalytic effect on the expression of pathogenic genes such as exoS, exoY, phzA1 and gacA and the production of PCN. On the one hand, the Pseudomonas aeruginosa can be changed. The results of this study have a better understanding of the function of GSH, the role of GSH in the process of Pseudomonas aeruginosa infection, and the pathogenic mechanism of P. aeruginosa.
【学位授予单位】：西北大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378
【引证文献】
相关期刊论文 前3条
1 朱帆;丁q，
本文编号：2480564
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2480564.html
最近更新
教材专著
