Hanson Mathew, Jordan Lorne D, Shipelskiy Yan, Newton Salete M, Klebba Phillip E
Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS, USA.
Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan, KS, USA
J Biomol Screen. 2016 Mar;21(3):316-22. doi: 10.1177/1087057115613788. Epub 2015 Oct 30.
The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8-1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria.
依赖TonB的革兰氏阴性菌外膜蛋白FepA可将铁载体肠杆菌素铁（FeEnt）主动转运至周质中。我们开发了一种高通量筛选（HTS）测定法，通过监测FeEnt结合时发生的荧光猝灭以及随后细菌通过转运将其从溶液中耗尽时荧光的恢复，来观察活的大肠杆菌中通过FepA摄取FeEnt的情况。我们优化了标记和光谱方法，以筛选通过外膜的依赖TonB的铁摄取抑制剂。该测定法就像一个分子开关，在存在TonB活性时开启，在不存在时关闭。它可在96孔微量滴定板中，在多种条件下发挥作用，Z因子为0.8 - 1.0。依赖TonB的铁转运是能量依赖型的，该测定法可轻松检测到代谢抑制剂羰基氰化物间氯苯腙、2,4 - 二硝基苯酚、叠氮化物、氰化物和砷酸盐对FeEnt摄取的抑制作用。由于铁的获取是细菌致病性的一个决定因素，用这种方法进行高通量筛选可能会鉴定出阻断TonB功能的抑制剂，并构成针对革兰氏阴性菌引起的传染病的新型疗法。
