铜绿假单胞菌新基因PA0058插入失活对氨基糖苷类抗生素耐药性的影响

Effects of insertional inactivation of novel gene PA0058 on the aminoglycoside resistance in Pseudomonas aeruginosa

摘要:

【目的】铜绿假单胞菌是一种重要的条件致病菌, 临床上常引起难治性和顽固性感染, 随着各种抗生素的广泛使用, 该菌对多种抗生素呈现耐药性, 研究其耐药性机理有着重要意义。【方法】以一株临床分离株Pseudomonas aeruginosa PA68作为出发菌株, 应用人工Mu转座技术构建突变文库并从中筛选得到一株对链霉素抗性明显增强的菌株M122, 并对突变株M122进行测序分析及表型检测。通过Southern杂交实验证实转座子是否为单拷贝插入, 对突变株M122的基因表达谱与野生型PA68菌株进行对比分析。【结果】确定了Mu转座子在M122基因组上为单拷贝插入, 插入位点为基因PA0058的第214 bp处。对M122进行表型检测, 发现其对多种氨基糖苷类抗生素的耐药性均得到增强, 通过转入携带完整基因PA0058的表达质粒可以使突变株M122的耐药性有所降低, 利用同源重组的方法, 在模式菌株P. aeruginosa PAK中进行PA0058基因敲除, 得到的敲除株具有链霉素耐药性升高的表型。基因PA0058的缺失引起多种基因表达水平改变, 尤其是katB、ahpC、ahpF等抗氧化酶基因转录表达显著增高。【结论】首次发现铜绿假单胞菌PA0058基因的插入失活提高了细菌对氨基糖苷类抗生素的耐药性, 且导致突变株M122中抗氧化酶基因转录表达水平的上调。

Abstract:

[Objective] Pseudomonas aeruginosa is a typical opportunistic pathogen which often causes serious and persistent infections in hospitals. As antibiotics have been used extensively and constantly, P. aeruginosa developing higher multi-drug resistance. Hence, investigating the mechanisms of antibiotic resistance in P. aeruginosa has significant clinical value. [Methods] A clinical isolated strain PA68 was adopted to construct a Mu transposon insertion mutant library. One mutant strain named M122 with abnormally high streptomycin resistance was isolated from the library. Southern blotting confirmed that the insertions had occurred as single event. DNA microarray was used to analyze the genomic expression in M122. [Results] Gene cloning and sequencing indicated that the alteration of M122 phenotype was due to the insertional activation of a novel gene PA0058. Mutation of PA0058 gene resulted in a higher resistance to several kinds of aminoglycoside antibiotics. After transforming a plasmid containing gene PA0058 into M122, the resistance phenotype restored partially. To exclude the strain specific, gene PA0058 was knocked-out in P. aeruginosia PAK. The results indicated that the mutation of gene PA0058 causes multiple gene expression change in P. aeruginosa, especially the significantly up-regulated antioxidant enzyme genes. [Conclusion] This is the first time the gene PA0058 was identified and reported to be involved in upward aminoglycoside resistance in P. aeruginosa.