Chen Shuzhen, Cao Huiluo, Xu Zirui, Huang Jiahui, Liu Zhiqing, Li Ting, Duan Cheng, Wu Weiyan, Wen Yongqi, Zhang Lian-Hui, Xu Zeling
Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Integrative Microbiology Research Centre, South China Agricultural University , Guangzhou, China.
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong , Hong Kong, China.
Microbiol Spectr. 2023 Aug 30;11(5):e0112323. doi: 10.1128/spectrum.01123-23.
has abundant signaling systems that exquisitely control its antibiotic resistance in response to different environmental cues. Understanding the regulation of antibiotic resistance will provide important implications for precise antimicrobial interventions. However, efficient genetic tools for functional gene characterizations are sometimes not available, particularly, in clinically isolated strains. Here, we established a type I-F CRISPRi (CSYi) system for programmable gene silencing. By incorporating anti-CRISPR proteins, this system was even applicable to bacterial hosts encoding a native type I-F CRISPR-Cas system. With the newly developed gene-silencing system, we revealed that the response regulator CzcR from the zinc (Zn)-responsive two-component system CzcS/CzcR is a repressor of efflux pumps MexAB-OprM and MexGHI-OpmD, which inhibits the expression of both operons by directly interacting with their promoters. Repression of MexAB-OprM consequently increases the susceptibility of to multiple antibiotics such as levofloxacin and amikacin. Together, this study provided a simple approach to study gene functions, which enabled us to unveil the novel role of CzcR in modulating efflux pump genes and multidrug resistance in . IMPORTANCE is a ubiquitous opportunistic pathogen frequently causing chronic infections. In addition to being an important model organism for antibiotic-resistant research, this species is also important for understanding and exploiting CRISPR-Cas systems. In this study, we established a gene-silencing system based on the most abundant type I-F CRISPR-Cas system in this species, which can be readily employed to achieve targeted gene repression in multiple bacterial species. Using this gene-silencing system, the physiological role of Zn and its responsive regulator CzcR in modulating multidrug resistance was unveiled with great convenience. This study not only displayed a new framework to expand the abundant CRISPR-Cas and anti-CRISPR systems for functional gene characterizations but also provided new insights into the regulation of multidrug resistance in and important clues for precise anti-pseudomonal therapies.
具有丰富的信号系统，可根据不同的环境线索精确控制其抗生素耐药性。了解抗生素耐药性的调控将为精准抗菌干预提供重要启示。然而，用于功能基因表征的高效遗传工具有时并不存在，特别是在临床分离菌株中。在此，我们建立了一种用于可编程基因沉默的I-F型CRISPRi（CSYi）系统。通过整合抗CRISPR蛋白，该系统甚至适用于编码天然I-F型CRISPR-Cas系统的细菌宿主。利用新开发的基因沉默系统，我们发现锌（Zn）响应双组分系统CzcS/CzcR中的响应调节因子CzcR是外排泵MexAB-OprM和MexGHI-OpmD的阻遏物，它通过直接与其启动子相互作用来抑制这两个操纵子的表达。对MexAB-OprM的抑制作用因此增加了对多种抗生素如左氧氟沙星和阿米卡星的敏感性。总之，本研究提供了一种研究基因功能的简单方法，使我们能够揭示CzcR在调节外排泵基因和多重耐药性中的新作用。重要性：铜绿假单胞菌是一种普遍存在的机会致病菌，经常引起慢性感染。除了是抗生素耐药性研究的重要模式生物外，该物种对于理解和利用CRISPR-Cas系统也很重要。在本研究中，我们基于该物种中最丰富的I-F型CRISPR-Cas系统建立了一个基因沉默系统，该系统可很容易地用于在多种细菌物种中实现靶向基因抑制。利用这个基因沉默系统，锌及其响应调节因子CzcR在调节多重耐药性中的生理作用被非常方便地揭示出来。本研究不仅展示了一个扩展丰富的CRISPR-Cas和抗CRISPR系统用于功能基因表征的新框架，还为铜绿假单胞菌多重耐药性的调控提供了新见解以及精准抗假单胞菌治疗的重要线索。
