Cryoelectron Microscopy Structures of AdeB Illuminate Mechanisms of Simultaneous Binding and Exporting of Substrates.

Christopher E Morgan,Anhthu Trinh,Meng Cui,Chih-Chia Su,Inga V Leus,Przemyslaw Glaza,Edward W Yu,Helen I Zgurskaya,Steven J Projan

doi:10.1128/mbio.03690-20

Christopher E Morgan, Anhthu Trinh + Show 7 more

Open Access

https://doi.org/10.1128/mbio.03690-20

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Abstract

ABSTRACTAcinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide. Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. The best-characterized multidrug efflux system in A. baumannii is the prevalent Acinetobacter drug efflux B (AdeB) pump, which is a member of the resistance-nodulation-cell division (RND) superfamily. Here, we report six structures of the trimeric AdeB multidrug efflux pump in the presence of ethidium bromide using single-particle cryoelectron microscopy (cryo-EM). These structures allow us to directly observe various novel conformational states of the AdeB trimer, including the transmembrane region of trimeric AdeB can be associated with form a trimer assembly or dissociated into “dimer plus monomer” and “monomer plus monomer plus monomer” configurations. We also discover that a single AdeB protomer can simultaneously anchor a number of ethidium ligands and that different AdeB protomers can bind ethidium molecules simultaneously. Combined with molecular dynamics (MD) simulations, we reveal a drug transport mechanism that involves multiple multidrug-binding sites and various transient states of the AdeB membrane protein. Our data suggest that each AdeB protomer within the trimer binds and exports drugs independently.

Highlights

Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide
To confirm that substrates bind A. baumannii Acinetobacter drug efflux B (AdeB) with sufficient affinity to visualize via structural analysis, we first purified the full-length multidrug efflux pump and used the technique of fluorescence polarization to quantify the interactions of purified AdeB with ethidium bromide (Et) and rhodamine 6G (R6G)
We found that the MICs of Et in the efflux-deficient AbD3 cells were 4 to 8 mg/liter, but the MIC was increased to 32 to 64 mg/liter when these AbD3 cells were complemented with the plasmid-borne Acinetobacter drug efflux ABC (AdeABC) pump

Summary

Introduction

Acinetobacter baumannii is a Gram-negative pathogen that has emerged as one of the most highly antibiotic-resistant bacteria worldwide Multidrug efflux within these highly drug-resistant strains and other opportunistic pathogens is a major cause of failure of drug-based treatments of infectious diseases. Acinetobacter baumannii is an opportunistic Gram-negative pathogen that has emerged as one of the most problematic and highly antibiotic-resistant bacteria in the world This bacterial pathogen exhibits a high level of antimicrobial resistance (AMR) to a broad spectrum of agents [1, 2]. In A. baumannii, the best-characterized multidrug efflux system is the prevalent Acinetobacter drug efflux ABC (AdeABC) tripartite system [13] This efflux system is capable of mediating resistance to a broad spectrum of clinically relevant antimicrobial agents, such as aminoglycosides, tetracyclines, macrolides, b-lactams, fluoroquinolones, chloramphenicol, and trimethoprim [1, 13,14,15,16,17,18,19,20]. It has been demonstrated that the presence of adeC can elevate the levels of resistance to several antibiotics [22]

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