Fluorescence High-Throughput Screening for Inhibitors of TonB Action.
Fluorescence High-Throughput Screening for Inhibitors of TonB Action.
复制标题
TonB 作用抑制剂的荧光高通量筛选。
DOI：
10.1128/jb.00889-16
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发表时间：
2017
影响因子：
3.2
通讯作者：
Klebba,PhillipE
中科院分区：
文献类型：
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作者：
Nairn,BrittanyL;Eliasson,OliviaS;Hyder,DallasR;Long,NoahJ;Majumdar,Aritri;Chakravorty,Somnath;McDonald,Peter;Roy,Anuradha;Newton,SaleteM;Klebba,PhillipE
革兰氏阴性细菌通过 TonB 依赖性外膜转运蛋白 (TBDT) 获得铁铁载体。通过荧光光谱高通量筛选 (FLHTS)，我们鉴定了大肠杆菌 FepA (EcoFepA) 摄取 TonB 依赖性铁肠杆菌素 (FeEnt) 的抑制剂。在 17,441 种化合物的初步筛选中发现的 165 种抑制剂中，我们在二次测试中评估了 20 种：TonB 依赖性铁铁载体摄取和大肠杆菌素杀灭以及质子动力依赖性乳糖转运。在所有测试中，20 个主要命中中有 6 个抑制了 TonB 依赖性活性。比较它们对 [59Fe]Ent 和 [14C]乳糖积累的影响表明其中几种是质子离子载体，但依布硒啉和 ST0082990 两种化学品可能不是质子离子载体，可能会抑制 TonB-ExbBD。 FLHTS 对抗大肠杆菌的能力促使我们将其应用于鲍曼不动杆菌。我们鉴定了其FepA直系同源物（AbaFepA），删除并克隆了其结构基因，在其表面环中基因改造了8个Cys取代，用荧光素标记它们，并对鲍曼不动杆菌中FeEnt的摄取进行了荧光光谱观察。 AbaFepA 中的几个 Cys 取代（S279C、T562C 和 S665C）很容易被荧光标记，然后适合作为 FeEnt 转运的传感器。与在大肠杆菌中一样，该测试监测了 AbaFepA 对 TonB 依赖性 FeEnt 的吸收。在使用鲍曼不动杆菌的微量滴定格式中，FLHTS 产生的 Z' 因子为 0.6 至 0.8。这些数据验证了 FLHTS 策略针对甚至远亲革兰氏阴性细菌病原体的策略。总体而言，它发现了阻断 TonB 依赖性转运的药物，并显示出寻找对抗革兰氏阴性 CRE（碳青霉烯类耐药肠杆菌科）/ESKAPE（屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌）的化合物的潜力。 肠杆菌属）病原体。我们的结果表明，更大的化合物库中可能存在数百种此类化学物质。 重要性 革兰氏阴性细菌的抗生素耐药性促使人们努力寻找针对新靶标的新化合物。 CRE/ESKAPE 病原体是耐药细菌，包括鲍曼不动杆菌，它是呼吸机相关性肺炎和败血症的常见原因。我们对大肠杆菌进行了荧光高通量筛选 (FLHTS)，以寻找 TonB 依赖性铁转运的抑制剂，并针对鲍曼不动杆菌进行了测试，然后采用 FLHTS 技术来直接筛选鲍曼不动杆菌。该方法可扩展到其他耐药革兰氏阴性病原体。阻断 TonB 作用的化合物可能会干扰真核宿主获取铁，从而构成抑菌抗生素，防止微生物在人类和动物中定植。 FLHTS 方法可以识别针对革兰氏阴性菌的物种特异性和广谱药物。
Gram-negative bacteria acquire ferric siderophores through TonB-dependent outer membrane transporters (TBDT). By fluorescence spectroscopic hgh-throughput screening (FLHTS), we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake through Escherichia coli FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 in secondary tests: TonB-dependent ferric siderophore uptake and colicin killing and proton motive force-dependent lactose transport. Six of 20 primary hits inhibited TonB-dependent activity in all tests. Comparison of their effects on [59Fe]Ent and [14C]lactose accumulation suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, are likely not proton ionophores and may inhibit TonB-ExbBD. The facility of FLHTS against E. coli led us to adapt it to Acinetobacter baumannii. We identified its FepA ortholog (AbaFepA), deleted and cloned its structural gene, genetically engineered 8 Cys substitutions in its surface loops, labeled them with fluorescein, and made fluorescence spectroscopic observations of FeEnt uptake in A. baumannii. Several Cys substitutions in AbaFepA (S279C, T562C, and S665C) were readily fluoresceinated and then suitable as sensors of FeEnt transport. As in E. coli, the test monitored TonB-dependent FeEnt uptake by AbaFepA. In microtiter format with A. baumannii, FLHTS produced Z′ factors 0.6 to 0.8. These data validated the FLHTS strategy against even distantly related Gram-negative bacterial pathogens. Overall, it discovered agents that block TonB-dependent transport and showed the potential to find compounds that act against Gram-negative CRE (carbapenem-resistant Enterobacteriaceae)/ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Our results suggest that hundreds of such chemicals may exist in larger compound libraries.IMPORTANCEAntibiotic resistance in Gram-negative bacteria has spurred efforts to find novel compounds against new targets. The CRE/ESKAPE pathogens are resistant bacteria that include Acinetobacter baumannii, a common cause of ventilator-associated pneumonia and sepsis. We performed fluorescence high-throughput screening (FLHTS) against Escherichia coli to find inhibitors of TonB-dependent iron transport, tested them against A. baumannii, and then adapted the FLHTS technology to allow direct screening against A. baumannii. This methodology is expandable to other drug-resistant Gram-negative pathogens. Compounds that block TonB action may interfere with iron acquisition from eukaryotic hosts and thereby constitute bacteriostatic antibiotics that prevent microbial colonization of human and animals. The FLHTS method may identify both species-specific and broad-spectrum agents against Gram-negative bacteria.