Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus

Abstract

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with sub-micromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

Chemical Basis of Combination Therapy to Combat Antibiotic Resistance

“…Efflux pumps contribute to another type of intrinsic drug resistance in both Gram-negative and Gram-positive bacteria. ,,, Efflux pumps are protein complexes that can “pump out” exotoxins such as antimicrobial agents and toxins from bacteria, thereby reducing their intracellular concentration. Clinical resistance to several antibiotics has been associated with the action of efflux pumps in all ESKAPE pathogens. , Efflux pump protein complexes are classified into five groups based on their sequence homology, substrate specificity, supramolecular characteristics, and energy source (Figure A), namely (i) A TP- b inding c assette (ABC) efflux pumps, (ii) m ulti-drug a nd t oxic compound e xtrusion (MATE) efflux pumps, (iii) m ajor f acilitator s uperfamily (MFS) efflux pumps, (iv) s mall m ulti-drug r esistance (SMR) efflux pumps, and (v) r esistance n odulation-cell d ivision (RND) efflux pumps, which are the most prominent and clinically relevant efflux pumps.…”

CryoEM structure of QacA, an antibacterial efflux transporter from Staphylococcus aureus

“…The region of TM13-EL7 hairpin-TM14 helical bundle that is solvent exposed is also the site of interactions for the two ICabs used in the study. Unlike recent studies on NorA and NorC where the antibodies interact by inserting the CDR loops into the vestibule of the transporter and lock the transporter into an outward-open state 13,22 , the ICabs interact with EL7 at distinct locations and hold it in place resembling a “molecular bow-tie”. Surprisingly, despite the interactions with two ICabs, the efflux activity of QacA, measured in spheroplasts, remains unhampered.…”

“…While transporters like NorA, MdfA, and LmrP comprise 12 TM helices and belong to the DHA1 class of transporters [12][13][14] , QacA, LfrA, and SmvA comprise 14 TM helices and are part of the DHA2 family 9,15,16 .…”

Occurrence and Variability of the Efflux Pump Gene norA across the Staphylococcus Genus

“…In another recent study, Shang and colleagues describe alterations in the 277–297 polypeptide region that have a significant impact on NorA efflux activity and resistance to fluoroquinolones and could be partially responsible for the functional differences of the NorA EP in S. aureus , particularly the substitutions Val281Ile, Phe288Ile, and Asn290Asp, suggesting that the 277–297 region plays a major role in NorA conformational stabilization [ 29 ]. However, this region is significantly variable among staphylococcal NorA sequences analyzed in our study.…”