Deciphering the molecular mechanism of multidrug efflux by the bacterial anaerobic transporter MdtF

Abstract

Antimicrobial resistance is one of the biggest threats to global public health and is exacerbated by the prevalence of multidrug resistant bacteria. Bacteria can tolerate antibiotics and noxious compounds within physiologically challenging environments, including extreme acidity and nutrient and oxygen depletion. In these conditions, the proton motive force-driven efflux pump MdtEF from the resistance-nodulation-cell division superfamily is upregulated; however, the molecular mechanism underlying its function remains uncharacterised. In this thesis, three cryo-electron microscopy structures of the Escherichia coli multidrug transporter MdtF were elucidated within native-lipid nanodiscs, including the wildtype protein (WT MdtF), a single point mutant with an altered multidrug resistance phenotype (V610F MdtF), and an associated substrate-bound form (V610F R6G-MdtF). This V610F single point mutation in the drug binding pocket of MdtF was previously reported to increase susceptibility to certain drugs while conferring reduced susceptibility to others.

In combination with functional assays and molecular dynamics, distinct transmembrane conformational state transitions were discovered within MdtF that induce an enhanced engagement within its proton relay network and an altered allosteric drug transport mechanism. This elicits a moderate MdtF-mediated efflux rate under physiological conditions which is more efficient at acidic pH, a duality that has not been observed within other resistance-nodulation-cell division efflux systems. This may be significant in the context of the fitness cost and energy expense of MdtF, and other transporters, within the transient acidic and anaerobic microhabitats of the mammalian gut, where Escherichia coli colonise and infect. Moreover, substrate polyspecificity is likely determined by drug binding domain and channel conformational plasticity, analogous to its closely related, constitutively expressed counterpart, AcrB. Furthermore, the single point mutation V610F induced structural rearrangements in the porter domain, forming a hydrophobic cleft in the drug binding pocket that may rationalise its altered multidrug efflux profile. Together, these findings contribute to mounting evidence that conformer allostery underlies the molecular basis of efflux pump specificity and function, and that single point mutations can profoundly impact acquired multidrug resistance phenotypes by modifying conformational plasticity.

Collectively, these results offer mechanistic insights essential for understanding how MdtF mediates bacterial xenobiotic and toxin removal, in addition to its function in the acidic, nutrient-deficient environments encountered in the gastrointestinal tract. Although membrane proteins account for more than 60 % of drug targets, they are notoriously difficult to investigate due to low yield of purification and the requirement for a hydrophobic membrane mimetic in vitro. In this thesis, reproducible workflows for the purification and characterisation of MdtF within a native-like environment were established which is important in the wider context of membrane protein study. Furthermore, functional assays demonstrated that MdtF-mediated efflux is inhibited by the efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide. These results, coupled with optimised workflows to investigate MdtF dynamics by hydrogen deuterium exchange mass spectrometry developed in this thesis, establish a foundation for future efflux pump inhibitor design aimed at restoring the activity of ineffective antibiotics, propelling research, development, and optimism for an end to this urgent global public health crisis.

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Identifiers

ORCID for Ryan James Lawrence: orcid.org/0000-0003-1232-3627

ORCID for Eamonn Reading: orcid.org/0000-0001-8219-0052

ORCID for Andrew Lawrence: orcid.org/0000-0002-5853-5409

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