Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong Special Administrative Region, China.
State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong Special Administrative Region, China.
Appl Environ Microbiol. 2024 Aug 21;90(8):e0071724. doi: 10.1128/aem.00717-24. Epub 2024 Jul 17.
Mechanistic investigations are of paramount importance in elucidating the modes of action of antibiotics and facilitating the discovery of novel drugs. We reported a luciferase-based reporter system using bacterial cells to unveil mechanisms of antimicrobials targeting transcription and translation. The reporter gene encoding NanoLuciferase (NanoLuc) was integrated into the genome of the Gram-positive model organism, , to generate a reporter strain BS2019. Cellular transcription and translation levels were assessed by quantifying the amount of mRNA as well as the luminescence catalyzed by the enzyme NanoLuc. We validated this system using three known inhibitors of transcription (rifampicin), translation (chloramphenicol), and cell wall synthesis (ampicillin). The reporter strain BS2019 successfully revealed a decline in expression by rifampicin and NanoLuc enzyme activity by chloramphenicol, while ampicillin produced no observable effect. The assay was employed to characterize a previously discovered bacterial transcription inhibitor, CUHK242, with known antimicrobial activity against drug-resistant . Production of mRNA in our reporter BS2019 was suppressed in the presence of CUHK242, demonstrating the usefulness of the construct, which provides a simple way to study the mechanism of potential antibiotic candidates at early stages of drug discovery. The reporter system can also be modified by adopting different promoters and reporter genes to extend its scope of contribution to other fields of work.
Discovering new classes of antibiotics is desperately needed to combat the emergence of multidrug-resistant pathogens. To facilitate the drug discovery process, a simple cell-based assay for mechanistic studies is essential to characterize antimicrobial candidates. In this work, we developed a luciferase-based reporter system to quantify the transcriptional and translational effects of potential compounds and validated our system using two currently marketed drugs. Reporter strains generated in this study provide readily available means for identifying bacterial transcription inhibitors as prospective novel antibacterials. We also provided a series of plasmids for characterizing promoters under various conditions such as stress.
在阐明抗生素的作用机制和促进新型药物发现方面，机制研究至关重要。我们报告了一种基于荧光素酶的报告系统，该系统使用细菌细胞揭示了针对转录和翻译的抗菌药物的作用机制。报告基因编码纳米荧光素酶（NanoLuc）被整合到革兰氏阳性模式生物的基因组中，以生成报告菌株 BS2019。通过定量 mRNA 的量以及酶 NanoLuc 催化的发光来评估细胞转录和翻译水平。我们使用三种已知的转录抑制剂（利福平）、翻译抑制剂（氯霉素）和细胞壁合成抑制剂（氨苄西林）验证了该系统。报告菌株 BS2019 成功地揭示了利福平导致的 表达下降和氯霉素导致的 NanoLuc 酶活性下降，而氨苄西林则没有观察到明显的效果。该测定法用于表征一种先前发现的具有抗药性的细菌转录抑制剂 CUHK242，该抑制剂具有已知的抗微生物活性。在存在 CUHK242 的情况下，我们报告菌株 BS2019 中的 mRNA 产生受到抑制，证明了该构建体的有用性，该构建体提供了一种在药物发现的早期阶段研究潜在抗生素候选物作用机制的简单方法。该报告系统还可以通过采用不同的启动子和报告基因进行修改，以将其应用范围扩展到其他工作领域。
迫切需要发现新的抗生素类别来对抗多药耐药病原体的出现。为了促进药物发现过程，需要一种简单的基于细胞的机制研究测定法来对抗菌候选物进行特征描述。在这项工作中，我们开发了一种基于荧光素酶的报告系统，用于量化潜在化合物的转录和翻译效应，并使用两种当前市售药物验证了我们的系统。本研究中生成的报告菌株为鉴定细菌转录抑制剂作为潜在新型抗菌药物提供了现成的手段。我们还提供了一系列质粒，用于在各种条件下（如应激）对启动子进行特征描述。
