Rapid Inhibition Profiling in Bacillus subtilis to Identify the Mechanism of Action of New Antimicrobials.
Rapid Inhibition Profiling in Bacillus subtilis to Identify the Mechanism of Action of New Antimicrobials.
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DOI：
10.1021/acschembio.5b01050
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发表时间：
2016-08-19
影响因子：
4
通讯作者：
Pogliano K
中科院分区：
文献类型：
--
作者：
Lamsa A;Lopez-Garrido J;Quach D;Riley EP;Pogliano J;Pogliano K
抗生素耐药性的增加已成为一个重大的公共卫生危机。迫切需要具有新型作用机制（MOA）的新型抗菌剂。我们以前开发了一种方法，细菌细胞学分析（BCP），它利用荧光显微镜快速识别抗菌化合物的MOA。BCP基于我们的发现，即用影响不同代谢途径的抗生素处理的细胞产生不同的细胞学特征，提供可用于确定化合物MOA的定量信息。在这里，我们描述了一个系统，快速抑制分析（RIP），用于创建新的抗生素靶点的细胞学概况，目前没有化学抑制剂。RIP由靶蛋白的快速、可诱导的降解组成，随后是BCP。我们证明，降解枯草芽孢杆菌中DNA复制、转录、脂肪酸生物合成和肽聚糖生物合成的主要代谢途径中的必需蛋白质迅速产生与针对相同途径的抗菌剂密切匹配的细胞学特征。此外，RIP和针对脂肪酸生物合成中不同步骤的抗生素可以彼此区分。我们利用RIP和BCP表明，四种非甾体抗炎抗生素的抗菌MOA与基于体外数据提出的不同。RIP是一种通用的方法，将扩展我们对与灭活必需细菌酶相关的表型的知识，从而允许筛选抑制新的必需靶标的分子。
Increasing antimicrobial resistance has become a major public health crisis. New antimicrobials with novel mechanisms of action (MOA) are desperately needed. We previously developed a method, bacterial cytological profiling (BCP), which utilizes fluorescence microscopy to rapidly identify the MOA of antimicrobial compounds. BCP is based upon our discovery that cells treated with antibiotics affecting different metabolic pathways generate different cytological signatures, providing quantitative information that can be used to determine a compound’s MOA. Here, we describe a system, rapid inhibition profiling (RIP), for creating cytological profiles of new antibiotic targets for which there are currently no chemical inhibitors. RIP consists of the fast, inducible degradation of a target protein followed by BCP. We demonstrate that degrading essential proteins in the major metabolic pathways for DNA replication, transcription, fatty acid biosynthesis, and peptidoglycan biogenesis in Bacillus subtilis rapidly produces cytological profiles closely matching that of antimicrobials targeting the same pathways. Additionally, RIP and antibiotics targeting different steps in fatty acid biosynthesis can be differentiated from each other. We utilize RIP and BCP to show that the antibacterial MOA of four nonsteroidal anti-inflammatory antibiotics differs from that proposed based on in vitro data. RIP is a versatile method that will extend our knowledge of phenotypes associated with inactivating essential bacterial enzymes and thereby allow for screening for molecules that inhibit novel essential targets.