College of Biotechnology and Bioengineering, Zhejiang University of Technologygrid.469325.f, Hangzhou, China.
Appl Environ Microbiol. 2022 Sep 22;88(18):e0084622. doi: 10.1128/aem.00846-22. Epub 2022 Aug 30.
There is an urgent need to develop novel antibiotics since antibiotic resistance is an increasingly serious threat to global public health. Whole-cell biosensors are one of the promising strategies for new antibiotic discovery. The peptidoglycan (PG) of the bacterial cell wall is one of the most important targets for antibiotics. However, the biosensors for the detection of PG-targeting antibiotics in Gram-negative bacteria have not been developed, mainly because of the lack of the regulatory systems that sense and respond to PG stress. Recently, we identified a novel two-component signal transduction system (PghKR) that is responsible for sensing and responding to PG damage in the Gram-negative bacterium Shewanella oneidensis. Based on this system, we developed biosensors for the detection of PG-targeting antibiotics. Using ampicillin as an inducer for PG stress and the bacterial luciferase LuxCDABE as the reporter, we found that the PghKR biosensors are specific to antibiotics targeting PG synthesis, including β-lactams, vancomycin, and d-cycloserine. Deletion of genes encoding PG permease AmpG and β-lactamase BlaA improves the sensitivity of the biosensors substantially. The PghKR biosensor in the background of Δ is also functional on agar plates, providing a simple method for screening bacteria that produce PG-targeting antibiotics. The growing problem of antibiotic resistance in Gram-negative bacteria urgently needs new strategies so that researchers can develop novel antibiotics. Microbial whole-cell biosensors are capable of sensing various stimuli with a quantifiable output and show tremendous potential for the discovery of novel antibiotics. As the Achilles' heel of bacteria, the synthesis of the peptidoglycan (PG) is targeted by many antibiotics. However, the regulatory systems that sense and respond to PG-targeting stress in Gram-negative bacteria are reported rarely, restricting the development of biosensors for the detection of PG-targeting antibiotics. In this study, we developed a highly sensitive and specific biosensor based on a novel two-component system in the Gram-negative bacterium Shewanella oneidensis that is responsible for the sensing and responding to PG stress. Our biosensors have great potential for discovering novel antibiotics and determining the mode of action of antibiotics.
由于抗生素耐药性对全球公共健康构成日益严重的威胁，因此迫切需要开发新型抗生素。全细胞生物传感器是新抗生素发现的一种很有前途的策略。细菌细胞壁的肽聚糖 (PG) 是抗生素的最重要靶标之一。然而，革兰氏阴性菌中用于检测 PG 靶向抗生素的生物传感器尚未开发，主要是因为缺乏感应和响应 PG 应激的调节系统。最近，我们鉴定了一种新型的双组分信号转导系统（PghKR），该系统负责感应和响应革兰氏阴性菌希瓦氏菌 Shewanella oneidensis 中的 PG 损伤。基于该系统，我们开发了用于检测 PG 靶向抗生素的生物传感器。我们使用氨苄青霉素作为 PG 应激诱导物，并用细菌荧光素酶 LuxCDABE 作为报告基因，发现 PghKR 生物传感器特异性地检测到靶向 PG 合成的抗生素，包括β-内酰胺类抗生素、万古霉素和 D-环丝氨酸。缺失编码 PG 通透酶 AmpG 和β-内酰胺酶 BlaA 的基因可显著提高生物传感器的灵敏度。在 Δ 背景下的 PghKR 生物传感器在琼脂平板上也具有功能，为筛选产生 PG 靶向抗生素的细菌提供了一种简单的方法。革兰氏阴性菌中抗生素耐药性日益严重的问题迫切需要新的策略，以便研究人员能够开发新型抗生素。微生物全细胞生物传感器能够感应各种刺激并产生可量化的输出，在新型抗生素的发现方面具有巨大潜力。作为细菌的阿喀琉斯之踵，肽聚糖 (PG) 的合成是许多抗生素的靶标。然而，感应和响应革兰氏阴性菌中 PG 靶向应激的调节系统很少有报道，这限制了用于检测 PG 靶向抗生素的生物传感器的开发。在这项研究中，我们在革兰氏阴性菌希瓦氏菌 Shewanella oneidensis 中开发了一种基于新型双组分系统的高度敏感和特异的生物传感器，该系统负责感应和响应 PG 应激。我们的生物传感器在发现新型抗生素和确定抗生素作用模式方面具有很大的潜力。
