Harnessing the native CRISPR-cas system for genome editing and antimicrobial research in multidrug resistant pseudomonas aeruginosa

Abstract

Genetic manipulation is a leading technology that revolutionizes the development of life sciences. One of the major threats in the current biomedicine is antimicrobial resistance (AMR), especially in the clinically significant “ESKAPE” organisms. A readily applicable and efficient genome-editing technology is imperative for molecular dissection of resistance determinants and development of anti-resistance interventions. While the recently developed, heterologous CRISPR-Cas9 system is broadly employed in the genome editing in mammalian cells, its application in bacteria is frequently hindered due to its cytotoxicity and the vast diversity of DNA homeostasis in bacteria. Pseudomonas aeruginosa is a prevalent and pernicious pathogen with extraordinary antibiotic and multidrug resistance. In this study, I use a clinically isolated, paradigm XDR (extensive drug-resistant) strain PA154197 to explore new genome-editing methodology and its exploitations in the AMR research.

Bioinformatics revealed that 67% of sequenced P. aeruginosa genomes contain native CRISPR-Cas systems with more than 90% of them belonging to the subtype I-E or I-F. Whether these native CRISPR-Cas systems can be harnessed for genome editing and antimicrobial research in the non-model, clinical P. aeruginosa remains unexplored. Herein, a native type I-F CRISPR-Cas-mediated genome-editing methodology is first developed in PA154197. Various genetic manipulations, including precise gene deletion, point mutation, in situ tagging etc. are achieved efficiently through a single transformation of a programmable editing plasmid pAY5235 containing a self-targeting mini-CRISPR, a repair donor and an antibiotic-luminescence dual selection marker. A two-step In-Del approach involving insertion and subsequent deletion of a tag nearby the desired editing site which lacks effective PAMs is further devised to achieve desired editing. This approach principally allows any kind of non-lethal genetic manipulations in the strain.

PA154197 is defined as an XDR strain because the only antipseudomonal antibiotic classes it remains susceptible to are aminoglycoside and the cyclic non-ribosomal polypeptides. Powered by bioinformatic analyses and the native CRISPR-Cas-mediated genome-editing methodology I developed, reverse mutations of the genes identified by previous transcriptome analysis to be potential resistance determinants are constructed in PA154197. I found that over-production of the MexAB-OprM and MexEF-OprN multidrug efflux pumps, decreased production of the OprD porin, and mutations in the DNA gyrase gene gyrA are responsible for the high resistance profile of PA154197. Mutations in the upstream transcriptional regulators responsible for the altered expression of mexAB, mexEF and oprD, namely, the T226G mutation in mexR and an 8-bp deletion in mexT, are also identified. Unexpectedly, I found that over-production of another multidrug efflux pump MexGHI-OmpD and point mutation in the resistance gene parS have no effect on the resistance development of PA154197. Further analysis on combined mutations reveals the extensive synergy of MexAB-OprM and MexEF-OprN systems in expelling of fluoroquinolones, trimethoprim, and chloramphenicol. These results indicate the complex interplay of resistant mutations in shaping the XDR profile of clinical strains and the importance of targeted molecular investigations in AMR research.

Furthermore, I demonstrate that the newly developed efficient and precise native CRISPR-Cas-mediated genome-editing methodology can be applied in another type I-F CRISPR-containing clinical P. aeruginosa isolate, demonstrating the great potential of the developed methodology for functional genomic investigations in the “non-model” P. aeruginosa isolates and other pathogens.