Dissecting the molecular determinants of the major multidrug efflux pump transporter MexB towards its antibiotic and physiological substrates : the multidrug-resistant genotype PA154197 as a paradigm

Abstract

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa is imposing a global public health threat. The resistance-nodulation-cell division (RND) multidrug efflux pump MexAB-OprM is a major drug resistance determinant in the pathogen. This tripartite efflux pump actively expels drugs employing energy conferred by the proton relay among the D407, D408, and K940 residues in MexB. Moreover, the pump is found to participate in several physiological processes in P. aeruginosa, such as detoxification and quorum sensing (QS). However, the atomic determinants of MexB involved in these processes are poorly understood. This study aims to systematically investigate this outstanding question in the native genetic background of a prototype MDR genotype PA154197, in which MexAB-OprM is overexpressed and a native CRISPR-Cas-based, highly efficient genome editing platform has been established, enabling precise point mutations in the chromosomal mexB locus and whole-cell structure-activity analysis. To overcome the undesired effect of the whole gene (ΔmexB) mutation which often causes compensatory expression of other efflux genes in P. aeruginosa, I first constructed the MexB D407N D408N mutant as a MexB-inactive negative control and showed that this variant did not trigger the overexpression of other RND pump genes in PA154197. It is shown that this mutant displayed a higher production of QS-regulated virulence traits than the PA154197 parent. RT-qPCR, in vivo gene expression, and LC-MS/MS analyses revealed that the rhl and pqs QS systems were activated in the MexB D407N D408N mutant. These results confirmed the physiological role of MexB in the QS of P. aeruginosa. Structural studies have revealed that antibiotic transport by MexB follows three entry channels towards two substrate binding pockets, the proximal binding pocket (PBP) and the distal binding pocket (DBP). These two binding pockets are separated by a switch-loop encompassing the T613–S623 residues. In the second part of my study, three residue clusters, Q176-V177-F178-G179-S180, Q273-D274-S276-I277, and F615-F617-R620 within a radius of 4 Å from MexB bound substrates, were selected for alanine substitutions. A mexB mutant library encompassing sixteen alanine substitutions in the chromosomal locus of mexB is constructed using the native CRISPR-Cas-mediated genome editing for systematic structure-activity investigations. In the third part, molecular determinants for recognition towards antibiotics, physiological substrates, and efflux pump inhibitors were investigated. It is demonstrated that the side chains of F615-F617-R620 residues in the switch loop are required for expelling fluoroquinolone antibiotics, whereas extrusion of β-lactam antibiotics relied on the Q176-V177-F178-G179-S180 residue cluster lining the distal wall of the DBP groove. These combined with the in silico docking analysis showed that ciprofloxacin, levofloxacin, meropenem, carbenicillin, piperacillin, aztreonam, and tetracycline are MexB cave binders. In contrast, ceftazidime and ceftriaxone are indicated as MexB groove binders. Furthermore, the F178, Q273, F615, F617, R620, and F628 residues are found to play an important role in the physiological substrates transport. Lastly, examining the differential inhibition mechanisms of the MexB inhibitor PAβN showed that PAβN blocks fluoroquinolone and β-lactam through the F615-F617-R620 and Q176-V177-F178-G179-S180 residue cluster, respectively. In summary, this study substantiates the role of the MexAB-OprM pump in the QS process of P. aeruginosa and provides substrates recognition patterns of MexB at the atomic level. Since MexB is a genetically encoded, highly conserved drug resistance determinant in P. aeruginosa and in other Gram-negative bacteria, these studies will facilitate the development of novel antimicrobial interventions against a broad range of drug-resistant pathogens.