Abstract
Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.
Highlights
Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria
We use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to directly measure changes in AcrB structural dynamics owing to binding of the ciprofloxacin (CIP) antibiotic and the well-studied phenylalanine-arginine-βnaphthylamide (PAβN) efflux pump inhibitor (EPI)[5,7,15,29]
We reveal that the PAβN EPI restricts the intrinsic motions of the drug-binding pockets as part of its mechanism of action and is effective against both AcrB wildtype (AcrBWT) and AcrBG288D
Summary
Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. Energized by the proton-motive force, AcrB transports a broad variety of toxic substances, including antibiotics, outside of the cell through a channel formed by the periplasmic adaptor protein, AcrA, and outer membrane channel, TolC2,3 It is constitutively expressed in many pathogenic Gram-negative bacteria and, with its homologues forming the most clinically relevant pumps, has become a target for drug discovery to tackle multidrug resistance (MDR)[4,5,6,7]. We use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to directly measure changes in AcrB structural dynamics owing to binding of the ciprofloxacin (CIP) antibiotic and the well-studied phenylalanine-arginine-βnaphthylamide (PAβN) efflux pump inhibitor (EPI)[5,7,15,29] Investigating both wildtype AcrB (AcrBWT) and a recently discovered G288D mutation (AcrBG288D), uncovered in a posttherapy MDR clinical isolate of Salmonella Typhimurium[30], we were able to further understand the structural and functional consequences of substrate and inhibitor binding and clinically relevant mutation. We demonstrate that an EPI can dually bind a
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