铜绿假单胞菌dsbM基因相关研究
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摘要
铜绿假单胞菌(Pseudomonas aeruginosa，PA)，又称绿脓杆菌，广泛分布于自然界、人体的皮肤、肠道和上呼吸道中。铜绿假单胞菌是一种重要的条件致病菌，也是引起严重的医院内获得性感染的常见病原菌之一。该菌也常引发慢性支气管炎及囊性纤维化继发感染等疾病，是严重烧伤、创伤感染病人，囊性纤维化病人及晚期肿瘤患者死亡的重要原因之一，严重危害着人类的健康和生命。随着临床抗生素药物应用的日益广泛，多耐药铜绿假单胞菌的出现,给医院有效的抗感染治疗带来极大的困扰。因此，对其耐药机理的研究对于预防和治疗由铜绿假单胞菌引起的感染有重要的意义。
本文以一株卡那霉素敏感的临床分离株P. aeruginosa PA68为出发菌株，应用人工Mu转座复合物技术建立突变子库，从中筛选出一株对链霉素耐药性明显增强的菌株，命名为M122，通过基因克隆、核苷酸序列测定及分析，确定基因组上的Mu插入失活位点为PA0058基因的第214bp处，在Pseudomonasaeruginosa基因组数据库(www. pseudomonas.com)中，PA0058基因被定义为一个功能未知的，和已报道的基因没有同源性的新基因。本文以这个新基因为研究对象，通过大量的实验数据对PA0058基因的功能进行了系统、深入的探讨，并首次将其命名为dsbM基因。
对dsbM突变株M122的表型进行检测，发现其对多种氨基糖苷类抗生素的耐药性都得到增强。通过同源重组实验，在模式菌株P. aeruginosa PAK中敲除dsbM基因,得到的基因敲除株具有链霉素耐药性增高的表型，与Mu插入突变株M122一致，证实了dsbM基因在模式菌株中同样能够影响氨基糖苷类抗生素的耐药性。通过导入携带完整dsbM基因的表达载体可以使突变株M122对链霉素和庆大霉素的耐药性有所降低，表型部分回复至野生型水平。
本文中在大肠杆菌中诱导表达了dsbM基因，并对其表达产物DsbM蛋白进行了酶学活性的分析。发现DsbM蛋白能够催化二硫键的氧化、还原和异构，是一种新型的二硫键氧化还原酶。通过DNA微阵列技术得到了突变株M122的基因表达谱，与相同背景的野生型PA68菌株对比分析发现，dsbM基因的失活能够影响全基因组的表达，其表达产物DsbM可能是一个作用范围广泛的，具有重要功能的蛋白质。在表达量发生变化的基因中，参与主动运输、能量代谢功能的基因最多，并发现突变株中由于dsbM基因的失活，促使多种抗氧化酶的基因转录表达增高，其中包括oxyR调控系统调控的katB, ahpB和ahpCF。酵母双杂交实验证实DsbM蛋白与OxyR蛋白之间存在相互作用。
本文推测，二硫键氧化还原酶DsbM是通过OxyR抗氧化调节系统影响氨基糖苷类抗生素耐药性的。通过对铜绿假单胞菌耐药性相关基因的研究，能够更深入的认识细菌产生耐药性的机理和代谢途径，为新药物的设计和研发提供理论依据，最终能针对性地设计药物用以治疗和抑制铜绿假单胞菌所引发的感染。
Pseudomonas aeruginosa is a main opportunistic human pathogen in nosocomialinfections and its intrinsical resistance to antibiotics limits the disinfection efficacy.Its pathogenesis and antibiotic resistance are due to the presence of serials virulencefactors. As antibiotics have been used extensively and constantly, P. aeruginosareflects higher multidrug resistance, and the trend is keeping ascending.
In this study, a clinical isolated strain PA68was adopted to construct a mutantlibrary from which a drug resistant screen was carried out. One mutant strain withhigh streptomycin resistance was isolated and named M122. Gene cloning andsequencing indicated that the alteration of M122phenotype was due to the insertionalinactivation of the gene PA0058, which is a new gene in P. aeruginosa.
In this study, the bacteriostatic experiment in vitro indicates that M122hasabnormally high aminoglycoside resistance. After transforming a plasmid containingthe PA0058into M122, the resistance phenotype restored partially. To exclude thestrain specific, PA0058gene was knocked-out in P. aeruginosia PAK. It is the firsttime that gene PA0058was identified to be involved in upward aminoglycosideresistance in P. aeruginosa.
The gene PA0058encodes a234residues conserved protein of unknownfunction. We cloned and expressed this gene in Escherichia coli. The primarystructure analysis of PA0058demonstrates that it contains a CXXC motif, which isconserved in thiol:disulfide oxidoreductases. The tertiary structure prediction showsthis protein has a thioredoxin-like fold. The studies on biochemical characters ofpurified PA0058suggest that it has thiol:disulfide oxidising, reducing andisomerisation activities. The biochemical characterization suggests that PA0058is anovel disulfide oxidoreductase. Hence, the protein was designated as DsbM.
DNA microarray was used to analyze the genomic expression in M122. Theresults indicated that the mutation of gene PA0058causes multiple gene expressionchange in P. aeruginosa, especially the significantly up-regulated antioxidant enzymegenes. Microarray analysis of the M122mutant showed its unusual phenotype might be related to the bacterial antioxidant defense system mediated by the oxyR regulon.
The study on the functions of dsbM gene has important implications forunderstanding the mechanisms underlying aminoglycoside resistance in P. aeruginosa.It will helpful to prevent and cure the infection caused by P. aeruginosa.
本文以一株卡那霉素敏感的临床分离株P. aeruginosa PA68为出发菌株，应用人工Mu转座复合物技术建立突变子库，从中筛选出一株对链霉素耐药性明显增强的菌株，命名为M122，通过基因克隆、核苷酸序列测定及分析，确定基因组上的Mu插入失活位点为PA0058基因的第214bp处，在Pseudomonasaeruginosa基因组数据库(www. pseudomonas.com)中，PA0058基因被定义为一个功能未知的，和已报道的基因没有同源性的新基因。本文以这个新基因为研究对象，通过大量的实验数据对PA0058基因的功能进行了系统、深入的探讨，并首次将其命名为dsbM基因。
对dsbM突变株M122的表型进行检测，发现其对多种氨基糖苷类抗生素的耐药性都得到增强。通过同源重组实验，在模式菌株P. aeruginosa PAK中敲除dsbM基因,得到的基因敲除株具有链霉素耐药性增高的表型，与Mu插入突变株M122一致，证实了dsbM基因在模式菌株中同样能够影响氨基糖苷类抗生素的耐药性。通过导入携带完整dsbM基因的表达载体可以使突变株M122对链霉素和庆大霉素的耐药性有所降低，表型部分回复至野生型水平。
本文中在大肠杆菌中诱导表达了dsbM基因，并对其表达产物DsbM蛋白进行了酶学活性的分析。发现DsbM蛋白能够催化二硫键的氧化、还原和异构，是一种新型的二硫键氧化还原酶。通过DNA微阵列技术得到了突变株M122的基因表达谱，与相同背景的野生型PA68菌株对比分析发现，dsbM基因的失活能够影响全基因组的表达，其表达产物DsbM可能是一个作用范围广泛的，具有重要功能的蛋白质。在表达量发生变化的基因中，参与主动运输、能量代谢功能的基因最多，并发现突变株中由于dsbM基因的失活，促使多种抗氧化酶的基因转录表达增高，其中包括oxyR调控系统调控的katB, ahpB和ahpCF。酵母双杂交实验证实DsbM蛋白与OxyR蛋白之间存在相互作用。
本文推测，二硫键氧化还原酶DsbM是通过OxyR抗氧化调节系统影响氨基糖苷类抗生素耐药性的。通过对铜绿假单胞菌耐药性相关基因的研究，能够更深入的认识细菌产生耐药性的机理和代谢途径，为新药物的设计和研发提供理论依据，最终能针对性地设计药物用以治疗和抑制铜绿假单胞菌所引发的感染。
Pseudomonas aeruginosa is a main opportunistic human pathogen in nosocomialinfections and its intrinsical resistance to antibiotics limits the disinfection efficacy.Its pathogenesis and antibiotic resistance are due to the presence of serials virulencefactors. As antibiotics have been used extensively and constantly, P. aeruginosareflects higher multidrug resistance, and the trend is keeping ascending.
In this study, a clinical isolated strain PA68was adopted to construct a mutantlibrary from which a drug resistant screen was carried out. One mutant strain withhigh streptomycin resistance was isolated and named M122. Gene cloning andsequencing indicated that the alteration of M122phenotype was due to the insertionalinactivation of the gene PA0058, which is a new gene in P. aeruginosa.
In this study, the bacteriostatic experiment in vitro indicates that M122hasabnormally high aminoglycoside resistance. After transforming a plasmid containingthe PA0058into M122, the resistance phenotype restored partially. To exclude thestrain specific, PA0058gene was knocked-out in P. aeruginosia PAK. It is the firsttime that gene PA0058was identified to be involved in upward aminoglycosideresistance in P. aeruginosa.
The gene PA0058encodes a234residues conserved protein of unknownfunction. We cloned and expressed this gene in Escherichia coli. The primarystructure analysis of PA0058demonstrates that it contains a CXXC motif, which isconserved in thiol:disulfide oxidoreductases. The tertiary structure prediction showsthis protein has a thioredoxin-like fold. The studies on biochemical characters ofpurified PA0058suggest that it has thiol:disulfide oxidising, reducing andisomerisation activities. The biochemical characterization suggests that PA0058is anovel disulfide oxidoreductase. Hence, the protein was designated as DsbM.
DNA microarray was used to analyze the genomic expression in M122. Theresults indicated that the mutation of gene PA0058causes multiple gene expressionchange in P. aeruginosa, especially the significantly up-regulated antioxidant enzymegenes. Microarray analysis of the M122mutant showed its unusual phenotype might be related to the bacterial antioxidant defense system mediated by the oxyR regulon.
The study on the functions of dsbM gene has important implications forunderstanding the mechanisms underlying aminoglycoside resistance in P. aeruginosa.It will helpful to prevent and cure the infection caused by P. aeruginosa.
引文
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