【作者】 张亚妮；

【导师】 段康民；

【作者基本信息】 西北大学 ， 微生物学， 2009， 博士

【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。更多还原

【Abstract】 Pseudomonas aeruginosa is the third leading opportunistic pathogen in humans;it can cause serious nosocomial infections.Glutathione(GSH) is one of the most important intra-and extracellular antioxidants,abnormal levels of GSH play an important role in disease pathologies.Evidence shows many pulmonary diseases such as cystic fibrosis(CF) are associated with the lower levels of GSH in the epithelial lining fluid(ELF).GSH is present at high concentrations in normal respiratory ELF, but deficient in CF ELF.Oxidative injury inflicted by the major pathogen, Pseudomonas aeruginosa,in CF lungs is one of the causes in the disease manifestation.Therefore,inhaled GSH therapy has been used in clinical trials to increase the GSH levels in the ELF of CF patients.Studies revealed that the ameliorative effect of GSH against antibiotics-induced damage due to its antioxidant properties;however,GSH has been shown to protect bacterial pathogens against antibiotic treatments,reducing bacterial sensitivity to a number of antibiotics.To understand the conflicting effects of GSH and the relation with P.aeruginosa,we investigated the effect of GSH on the pathogenicity of P.aeruginosa and its sensitivity to different antibiotics.To study the relationship between GSH and the pathogenicity of P.aeruginosa,a promoters library of the virulence factor genes in P.aeruginosa based on the luxCDABE-reporter system was used.The levels of gene expression were monitored by measuring the light production of the luxCDABE reporters.Strains containing the reporters were treated with GSH or buthionine sulfoximine(BSO) and diethylmaleate (DEM) to examine the effect of GSH levels on the expression of virulence factor genes.Glutathione synthetase gene(gshB) mutant PAO1(△gshB) and glutathione reductase gene(gor) mutant PAO1(△gor) were also constructed to confirm the effect of GSH on virulence factor genes.The results indicate that the presence of GSH increased the expression of exoY、exoS、phzA1 and gacA,while no difference was observed with others virulence factor genes tested.PAO1(AgshB) produced markedly less pyocyanin(PCN) than the wild type strain PAOI(P＜0.01);Treating with GSH, the PCN production of PAOI(△gshB) was increased from 0.47μg/mL to 1.73μg/mL (P＜0.01),the PCN production of PAO1 increased from 1.3μg/mL to 1.9μg/mL(P＜0.05),this result indicates that GSH upregulates PCN production. Because exoY、exoS、phzA1 and gacA are related to quorum sensing systems,we investigated the relationship between GSH and QS,and the results indicated that the expression of the gshB and gor gene was downregulated by rhl system.Besides the virulence factor genes,the expression of MexXY-OprM was also decreased in PAOI(AgshB).It was restored to wild type levels when gshB was complemented, confirming the role of gshB.To test the effect of GSH on antibiotic sensitivity of P.aeruginosa,both disk fusion and MIC measurement were used.The results revealed that the presence of 3μg/μL of GSH decreased the sensitivity of P.aeruginosa against kanamycin, erythromycin,cefotaxime sodium,ciprofloxacin,carbenicillin,streptomycin,but interestingly,increased against tetracycline.The MICs against kanamycin was increased from 240μg/mL to 1600μg/mL,the MICs against tetracycline were decreased from 25μg/mL to 1.25μg/mL.The sensitivity to ampicillin and chloramphenicol remained unchanged.The effect of GSH or GSSG on antibiotic activity in PAO1(△gor) mutant was the same as in PAO1.These results indicated that effect of GSH on the sensitivity of P.aeruginosa to different antibiotics differs depending on the types of antibiotics.GSH altered antibiotic sensitivity seems to be unrelated to oxidative stress relief.In summary,this study demonstrates that GSH could increase the expression of exoY、exoS、phzA1、gacA and the production of PCN,and GSH could alter antibiotic sensitivity of P.aeruginosa to different antibiotics.Such effects have to be taken into consideration when GSH treatment is used in pulmonary disearse such as CF where P. aeruginosa infection is involved.These findings highlight the complexity of GSH effects on the host and as well as the pathogens,and uncover new relationship between GSH and the pathogenesis of P.aeruginosa.更多还原