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首页> 外文期刊>Antimicrobial agents and chemotherapy. >Streptococcus pneumoniae DNA gyrase and topoisomerase IV: overexpression, purification, and differential inhibition by fluoroquinolones.
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Streptococcus pneumoniae DNA gyrase and topoisomerase IV: overexpression, purification, and differential inhibition by fluoroquinolones.

机译：肺炎链球菌DNA促旋酶和拓扑异构酶IV：氟喹诺酮类药物的过度表达，纯化和差异抑制。

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Streptococcus pneumoniae gyrA and gyrB genes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones-ciprofloxacin, sparfloxacin, and clinafloxacin-which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity against S. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.
机译：已经在可诱导的T7启动子的控制下将指定DNA促旋酶亚基的肺炎链球菌gyrA和gyrB基因克隆到pET质粒载体中，并已在大肠杆菌中单独表达。通过一步镍螯合柱层析将可溶性97-kDa GyrA和72-kDa GyrB蛋白在其各自的C末端和N末端带有聚组氨酸标签，纯化至表观均质，并且不具有宿主大肠杆菌拓扑异构酶活性。等摩尔量的回旋酶亚基重建了ATP依赖的DNA超螺旋，其活性与大肠杆菌和金黄色葡萄球菌的回旋酶相当。平行地，肺炎链球菌拓扑异构酶IV ParC和ParE亚基在大肠杆菌中相似地表达，纯化为93-kDa和73-kDa蛋白质接近均一，并显示可产生有效的ATP依赖性DNA弛豫和DNA分解活性。使用纯化的酶，我们检查了三种范式氟喹诺酮类-环丙沙星，司帕沙星和克林沙星-先前的肺炎链球菌遗传研究表明，它们分别通过拓扑异构酶IV，回旋酶和两种酶优先发挥抑制作用。出乎意料的是，所有三种喹诺酮类药物在抑制纯化的拓扑异构酶IV方面都比促旋酶更有效，其中克林沙星显示出最大的抑制效力。此外，被测试的药物在稳定拓扑异构酶IV的可裂解复合物（相关的细胞毒性损伤）方面比在促旋酶方面至少高25倍，而克林沙星对任何一种酶的效力要高10到32倍，与其对肺炎链球菌有较高的活性。三种氟喹诺酮类药物在体外对拓扑异构酶IV的一致靶标偏好与遗传数据形成明显对比。我们用喹诺酮类杀死细菌的模型来解释这些结果，其中细胞因子可以调节靶标亲和力的作用来确定细胞毒性途径。

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