Native CRISPR-Cas-Mediated Genome Editing Enables Dissecting and Sensitizing Clinical Multidrug-Resistant P. aeruginosa

Cell Rep. 2019 Nov 5;29(6):1707-1717.e3. doi: 10.1016/j.celrep.2019.10.006. | PubMed

Zeling Xu¹, Ming Li², Yanran Li¹, Huiluo Cao¹, Lu Miao³, Zhaochao Xu³, Yusuke Higuchi⁴, Seiji Yamasaki⁴, Kunihiko Nishino⁴, Patrick C Y Woo⁵, Hua Xiang⁶, Aixin Yan⁷

1. School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
2. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
3. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
4. Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan.
5. Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
6. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. Electronic address: xiangh@im.ac.cn.
7. School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Electronic address: ayan8@hku.hk.

Abstract

Despite being fundamentally important and having direct therapeutic implications, the functional genomics of the clinical isolates of multidrug-resistant (MDR) pathogens is often impeded by the lack of genome-editing tools. Here, we report the establishment of a highly efficient, in situ genome-editing technique applicable in clinical and environmental isolates of the prototypic MDR pathogen P. aeruginosa by harnessing the endogenous type I-F CRISPR-Cas systems. Using this approach, we generate various reverse mutations in an epidemic MDR genotype, PA154197, and identify underlying resistance mechanisms that involve the extensive synergy among three different resistance determinants. Screening a series of "ancestor" mutant lines uncovers the remarkable sensitivity of the MDR line PA154197 to a class of small, cationic peptidomimetics, which sensitize PA154197 cells to antibiotics by perturbing outer-membrane permeability. These studies provide a framework for molecular genetics and anti-resistance drug discovery for clinically isolated MDR pathogens.

Presented By Zeling Xu