谷胱甘肽與銅綠假單胞菌致病性及抗生素抗性的關(guān)系研究
發(fā)布時(shí)間：2019-05-19 08:26
【摘要】： 銅綠假單胞菌(Pseudomonas aeruginosa)是病人在醫(yī)院發(fā)生感染的第三大條件致病菌,對(duì)宿主細(xì)胞引起的氧化傷害,是其導(dǎo)致病人感染死亡的原因之一。谷胱甘肽(glutathione,GSH)是細(xì)胞內(nèi)最重要的抗氧化劑之一,其含量變化與疾病的發(fā)生有著密切的關(guān)系。資料顯示許多肺部疾病如囊腫性纖維化(cysticfibrosis,CF)病人的呼吸道上皮分泌液(epithelial lining fluid,ELF)中,GSH的水平異常低下。由銅綠假單胞菌感染引起的氧化傷害也是導(dǎo)致CF病人癥候出現(xiàn)的主要原因之一。因此,在臨床實(shí)驗(yàn)中已有用吸入GSH的方法來提高CF病人ELF中的GSH水平。另據(jù)資料顯示,GSH一方面可以減輕抗生素作用細(xì)菌時(shí)對(duì)宿主細(xì)胞造成的氧化損害,一方面又消除了一些抗生素所引起的細(xì)菌細(xì)胞內(nèi)氧化壓力,保護(hù)細(xì)菌抵御抗生素的抑殺,降低細(xì)菌對(duì)一些抗生素的敏感性。為了了解GSH在細(xì)胞中這種功能上的矛盾以及和銅綠假單胞菌之間的關(guān)系,我們深入研究了GSH和銅綠假單胞菌致病性的關(guān)系以及對(duì)不同抗生素敏感性的影響。 為了研究GSH與銅綠假單胞菌致病性的關(guān)系,以熒光素酶基因操縱子luxCDABE發(fā)光報(bào)道基因構(gòu)建的致病性因子啟動(dòng)子庫為平臺(tái),外加不同濃度GSH或化學(xué)試劑耗竭細(xì)胞內(nèi)GSH,對(duì)啟動(dòng)子庫進(jìn)行篩選比較,依據(jù)luxCDABE報(bào)道子發(fā)光值的變化情況,尋找與GSH有關(guān)的致病性因子;構(gòu)建銅綠假單胞菌谷胱甘肽合成酶基因突變體PAO1(△gshB)和谷胱甘肽還原酶突變體PAO1(△gor),在分子水平進(jìn)一步確認(rèn)GSH和這些致病性因子的關(guān)系,分析GSH對(duì)銅綠假單胞菌致病性的影響。結(jié)果發(fā)現(xiàn),GSH對(duì)啟動(dòng)子庫中exoS、exoY、phzA1和gacA等致病性因子的表達(dá)有促進(jìn)作用,對(duì)其余測(cè)試過的致病性因子則沒有明顯影響;銅綠假單胞菌分泌的重要致病因子綠膿菌素(pyocyanin,PCN)的含量,在PAO1(△gshB)中遠(yuǎn)低于PAO1(P＜0.01);加入GSH后,PCN平均含量在PAO1(△gshB)中從0.47μg/mL提高到1.73μg/mL,差異十分顯著(P＜0.01),在PAO1中從1.3μg/mL提高到1.9μg/mL,差異顯著(P＜0.05),表明GSH對(duì)銅綠假單胞菌生產(chǎn)PCN有促進(jìn)作用。由于exoS、exoY、phzA1、gacA和PCN與群體感應(yīng)系統(tǒng)(Quorum-sensing,QS)有著密切的關(guān)系,我們對(duì)GSH和QS系統(tǒng)的關(guān)系進(jìn)行了相關(guān)研究,結(jié)果發(fā)現(xiàn),QS系統(tǒng)中rhl系統(tǒng)對(duì)gshB和gor基因有負(fù)調(diào)節(jié)作用。除了對(duì)以上致病性因子產(chǎn)生影響外,發(fā)現(xiàn)銅綠假單胞菌外排泵MexXY-OprM的表達(dá)在PAO1(△gshB)中顯著降低,并通過互補(bǔ)實(shí)驗(yàn)證實(shí)是由gshB基因的突變引起的,說明GSH對(duì)MexXY-OprM的表達(dá)有促進(jìn)作用。 為了研究GSH對(duì)抗生素抗菌活性的影響,利用瓊脂擴(kuò)散方法和最低抑菌濃度(MIC)方法,研究了GSH的存在引起的銅綠假單胞菌對(duì)不同抗生素敏感性的變化。結(jié)果發(fā)現(xiàn),在LB液體中添加3μg/μL的GSH,銅綠假單胞菌對(duì)卡那霉素、紅霉素、頭孢噻肟鈉、環(huán)丙沙星、羧芐青霉素和硫酸鏈霉素的敏感性降低,而對(duì)四環(huán)素的敏感性增加。銅綠假單胞菌對(duì)卡那霉素的MIC由240μg/mL增加為1600μg/mL,對(duì)四環(huán)素的MIC由25μg/mL降為1.25μg/mL,但是對(duì)氯霉素和氨芐青霉素并沒有明顯地影響。GSSG和GSH具有相同的功能,也可以改變PAO1對(duì)抗生素的敏感性。在PAO1(△gor)和PAO1(△gshB)兩個(gè)突變株中,GSSG和GSH同時(shí)可以影響抗生素的敏感性,其程度與野生型相同。上述結(jié)果顯示,GSH影響銅綠假單胞菌對(duì)不同抗生素的敏感性與抗生素的種類密切相關(guān),而與氧化型還是還原型谷光甘肽沒有關(guān)系,因此其作用可能不是通過改變細(xì)胞內(nèi)的氧化還原狀態(tài)而起作用的。 總之,GSH一方面對(duì)exoS、exoY、phzA1和gacA等致病性基因的表達(dá)和PCN的產(chǎn)生起到促進(jìn)作用,一方面可以改變銅綠假單胞菌對(duì)不同抗生素的敏感性。本研究的結(jié)果對(duì)于GSH生物功能的重新認(rèn)識(shí)、GSH在銅綠假單胞菌感染過程中所扮演的角色,以及銅綠假單胞菌的致病性機(jī)理研究等方面具有較大的理論價(jià)值和實(shí)際意義。
[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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378
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