Alanine Scanning Analysis of MexB in Multidrug-Resistant Pseudomonas aeruginosa Using the Native Type I-F CRISPR-Cas-Mediated Genome Editing.

The rising multidrug resistance (MDR) of the Gram-negative opportunistic pathogen Pseudomonas aeruginosa poses a global public health threat. One key resistance mechanism employed by this pathogen is the overexpression of the resistance-nodulation-cell division (RND) multidrug efflux transporters. MexAB-OprM, a prototype RND efflux pump, confers resistance to most conventional antibiotics except aminoglycosides. Molecules capable of inhibiting efflux pumps, i.e., efflux pump inhibitors (EPIs), have shown promise as therapeutic agents capable of restoring antibiotic efficacy against these superbugs by targeting MexB. Nevertheless, the structure-activity relationship governing substrate transport of MexB in the native genetic background of clinical MDR P. aeruginosa isolates is poorly characterized due to the lack of efficient genetic tools, hindering targeted EPIs development.In this chapter, we introduce a native Type I-F CRISPR-mediated precise genome editing technique that enables in situ alanine substitutions in MexB in clinical MDR P. aeruginosa genotypes. We outline detailed procedures to construct MexB alanine-substitution mutations and methods to examine the expression levels and efflux activities of the constructed MexB mutants. Methods to evaluate the effect of constructed alanine substitutions on antibiotic susceptibilities are also described. These methodologies are expected to provide advanced insights into the substrate recognition and selection mechanism of MexB in the native genetic background of clinical MDR P. aeruginosa isolates to facilitate the development of effective EPIs.