Abstract:
Antibiotic-resistant Enterobacterales pose a major threat to healthcare systems worldwide, necessitating the development of novel strategies to fight such hard-to-kill bacteria. One potential approach is to develop molecules that force bacteria to hyper-activate prodrug antibiotics, thus rendering them more effective. In the present work, we aimed to obtain proof-of-concept data to support that small molecules targeting transcriptional regulators can potentiate the antibiotic activity of the prodrug metronidazole (MTZ) against Escherichia coli under aerobic conditions. By screening a chemical library of small molecules, a series of structurally related molecules were identified that had little inherent antibiotic activity but showed substantial activity in combination with ineffective concentrations of MTZ. Transcriptome analyses, functional genetics, thermal shift assays, and electrophoretic mobility shift assays were then used to demonstrate that these MTZ boosters target the transcriptional repressor MarR, resulting in the upregulation of the marRAB operon and its downstream MarA regulon. The associated upregulation of the flavin-containing nitroreductase, NfsA, was then shown to be critical for the booster-mediated potentiation of MTZ antibiotic activity. Transcriptomic studies, biochemical assays, and electron paramagnetic resonance measurements were then used to show that under aerobic conditions, NfsA catalyzed 1-electron reduction of MTZ to the MTZ radical anion which in turn induced lethal DNA damage in E. coli. This work reports the first example of prodrug boosting in Enterobacterales by transcriptional modulators and highlights that MTZ antibiotic activity can be chemically induced under anaerobic growth conditions.
摘要:
抗生素耐药肠杆菌对全球医疗保健系统构成重大威胁,需要开发新的策略来对抗这种难以杀死的细菌。一种潜在的方法是开发迫使细菌过度激活前药抗生素的分子,从而使它们更有效。在目前的工作中,我们的目的是获得概念验证数据,以支持靶向转录调节因子的小分子可以增强前药甲硝唑(MTZ)在有氧条件下对大肠杆菌的抗生素活性.通过筛选小分子的化学文库,确定了一系列结构相关的分子,这些分子几乎没有固有的抗生素活性。但与无效浓度的MTZ组合显示出实质性活性。转录组分析,功能遗传学,然后使用热转移测定和电泳迁移率转移测定来证明这些MTZ增强剂靶向转录阻遏物MarR,导致marRAB操纵子及其下游MarA调节子上调。然后显示含黄素的硝基还原酶的相关上调;NfsA对于MTZ抗生素活性的加强介导的增强至关重要。转录组研究,然后使用生化测定和电子顺磁共振测量来表明在有氧条件下,NfsA催化MTZ的1电子还原为MTZ自由基阴离子,进而诱导大肠杆菌中的致命DNA损伤。这项工作报告了通过转录调节剂在肠杆菌中增强前药的第一个实例,并强调了MTZ抗生素活性可以在厌氧生长条件下化学诱导。