Substrate Specificity Checkpoints of the Multidrug Efflux Pump MexF from Pseudomonas aeruginosa

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Multidrug efflux pumps, particularly the resistance-nodulation-division (RND) superfamily, are critical in driving antibiotic resistance in *Pseudomonas aeruginosa*.
• The MexEF-OprN efflux system confers resistance to fluoroquinolones, trimethoprim, and chloramphenicol when overexpressed in clinical isolates.
• The inner-membrane RND transporter MexF within this system has a narrow substrate specificity, with underlying molecular mechanisms yet to be fully elucidated.

Purpose of the Study:

• To investigate the role of the Access pocket, a key recognition/binding site in MexF, in determining substrate specificity.
• To understand how mutations in specific residues affect the drug efflux capabilities and substrate spectrum of MexF.

Main Methods:

• Utilized a combination of experimental and computational approaches.
• Introduced mutations at key positions (D132, P136, G626, S729) in MexF.
• Assessed changes in resistance profiles and substrate specificity.
• Employed ensemble docking and contact frequency analyses to study ligand binding and translocation pathways.

Main Results:

• Mutations at D132, P136, G626, and S729 altered MexF's resistance profiles and substrate specificity in a residue- and substrate-dependent manner.
• Substitutions at P136 generally enhanced the efflux of various antibiotics, including numerous fluoroquinolones.
• Substitutions at S729 showed variable effects, either enhancing or significantly impairing MexF activity.
• Competition assays revealed overlapping binding sites and shared translocation paths for different antibiotic substrates.
• Computational analyses indicated that mutations remodel ligand binding preferences and influence transition to the Deep pocket for extrusion.

Conclusions:

• MexF is intrinsically optimized for trimethoprim-like compounds, but single residue substitutions can drastically broaden its substrate spectrum.
• The study highlights the importance of spatial occupancy and pathway integrity, in addition to static binding contacts, for efficient drug efflux by polyspecific transporters like MexF.
• Understanding these mechanisms is crucial for developing strategies to combat antibiotic resistance mediated by efflux pumps.