Li He, Quan Shu, He Wei
Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, East China University of Science and Technology, Shanghai, China.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai, China.
Front Chem. 2025 Jan 10;12:1528626. doi: 10.3389/fchem.2024.1528626. eCollection 2024.
Cyclic di-guanosine monophosphate (c-di-GMP) acts as a second messenger regulating bacterial behaviors including cell cycling, biofilm formation, adhesion, and virulence. Monitoring c-di-GMP levels is crucial for understanding these processes and designing inhibitors to combat biofilm-related antibiotic resistance. Here, we developed a genetically encoded biosensor, cdiGEBS, based on the transcriptional activity of the c-di-GMP-responsive transcription factor MrkH. Notably, cdiGEBS can detect both low and high cellular c-di-GMP levels, with a high fluorescence dynamic change of 23-fold. Moreover, it can detect subtle changes in c-di-GMP concentrations due to variations in the expression of c-di-GMP synthesis or degradation enzymes and can distinguish different synthesis activities among WspR mutants. These capabilities allow us to apply cdiGEBS for identifying new diguanylate cyclases and evaluating chemicals that modulate c-di-GMP levels, highlighting its potential as a high-throughput tool for screening inhibitors of c-di-GMP synthesis enzymes. Overall, cdiGEBS enhances the study of c-di-GMP-regulated functions and holds the potential for screening antimicrobials targeting c-di-GMP or its synthesis enzymes.
环二鸟苷单磷酸（c-di-GMP）作为第二信使，调节包括细胞周期、生物膜形成、黏附及毒力等细菌行为。监测c-di-GMP水平对于理解这些过程以及设计抑制剂以对抗生物膜相关的抗生素耐药性至关重要。在此，我们基于c-di-GMP响应转录因子MrkH的转录活性，开发了一种基因编码的生物传感器cdiGEBS。值得注意的是，cdiGEBS能够检测低水平和高水平的细胞内c-di-GMP，荧光动态变化高达23倍。此外，它能够检测由于c-di-GMP合成或降解酶表达变化而导致的c-di-GMP浓度的细微变化，并且能够区分WspR突变体之间不同的合成活性。这些能力使我们能够应用cdiGEBS来鉴定新的二鸟苷酸环化酶并评估调节c-di-GMP水平的化学物质，突出了其作为筛选c-di-GMP合成酶抑制剂的高通量工具的潜力。总体而言，cdiGEBS加强了对c-di-GMP调节功能的研究，并具有筛选靶向c-di-GMP或其合成酶的抗菌药物的潜力。
