耐甲氧西林金黄色葡萄球菌MRSA252辅酶A二硫化物还原酶缺失延缓苯唑西林的次级损伤效应  

Deficiency of coenzyme A disulfide reductase in methicillin-resistant Staphylococcus aureus MRSA252 delays the secondary damage of oxacillin

作　　者：杨萍[1,2] 刘蔷 蒋宇 孙兵兵[3] 杨俊杰[3] 李琦[4] 杨晟 陈代杰[5] Ping Yang;Qiang Liu;Yu Jiang;Bingbing Sun;Junjie Yang;Qi Li;Sheng Yang;Daijie Chen(School of Pharmacy,Fudan University,Shanghai 201203,China;Shanghai Institute of Pharmaceutical Industry,Shanghai 201203,China;Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200032,China;College of Life Sciences,Sichuan Normal University,Chengdu 610101,Sichuan Province,China;School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]复旦大学药学院,上海201203 [2]上海医药工业研究院,上海201203 [3]中国科学院上海生命科学研究院植物生理生态研究所,上海200032 [4]四川师范大学生命科学学院,四川成都610101 [5]上海交通大学药学院,上海200240

出　　处：《微生物学报》2019年第12期2296-2305,共10页Acta Microbiologica Sinica

基　　金：国家自然科学基金(81573329,81872775,21825804);上海市自然科学基金(18ZR1446800,18ZR1446500)~~

摘　　要：【目的】耐甲氧西林金黄色葡萄球菌在苯唑西林作用下,其辅酶A二硫化物还原酶表达上调2.3倍。本文研究苯唑西林对该酶缺失的金黄色葡萄球菌的杀菌效应。【方法】利用同源重组双交换技术对金黄色葡萄球菌进行基因敲除,并用质粒p OS1构建回补株;采用分光光度法检测菌株体外增殖能力;以时间-杀菌法考察苯唑西林对菌株杀菌效应;以2’,7’-二氯荧光黄双乙酸盐为探针检测胞内活性氧水平。【结果】辅酶A二硫化物还原酶基因敲除株较亲株生长缓慢(P<0.05);20倍MIC浓度苯唑西林下敲除株的时间-杀菌曲线及胞内活性氧水平与亲株无显著性差异,5倍MIC浓度下敲除株的致死速率及胞内活性氧水平均较亲株下降。【结论】在较低浓度苯唑西林作用下,辅酶A二硫化物还原酶基因缺失降低胞内活性氧水平,减小杀菌速率,延缓次级损伤效应。[Objective] The expression of coenzyme A disulfide reductase in methicillin-resistant Staphylococcus aureus was up-regulated by 2.3 folds when the strain was cultured with oxacillin. Here, the bactericidal effect of oxacillin on coenzyme A disulfide reductase deficient Staphylococcus aureus was studied. [Methods] Homologous recombination mediated double-exchange techniques were used to delete genes in Staphylococcus aureus, whereas pOS1 plasmid was used to construct the complemented strain. Spectrophotometry was used to detect the proliferation ability of the strains in vitro. The bactericidal effect of oxacillin on the strains was studied by time-kill method, and 2’,7’-dichlorodihydrofluorescein diacetate was used as a probe to detect intracellular reactive oxygen species levels. [Results] Coenzyme A disulfide reductase gene deleted strain grew more slowly than the parental strain(P<0.05). Under the action of oxacillin, the time-kill curve and intracellular reactive oxygen species level of the knockout strains were slightly different from that of the parental strains at 20 times MIC concentration, and the lethal rate and intracellular reactive oxygen species level of coenzyme A disulfide reductase gene deleted strains were lower than that of the parental strains at 5 times MIC concentrations. [Conclusion] At lower concentrations of oxacillin, the deletion of coenzyme A disulfide reductase gene reduced intracellular ROS levels, and the bactericidal rate, meanwhile the secondary damage was delayed.

关 键 词：金黄色葡萄球菌 基因编辑 辅酶A二硫化物还原酶 活性氧 次级损伤 

分 类 号：R44[医药卫生—诊断学]