Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

Abstract

Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

A Strain-Specific Inhibitor of Receptor-Bound HIV-1 Targets a Pocket near the Fusion Peptide

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A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids

“…The purified AcrB protein was then concentrated to a final monomeric concentration of 10 μM in buffer containing 20 mM Na-HEPES (pH 7.5) in 0.05% DDM. Similar protein purification procedures have been used to elucidate structure-function of RND transporters, including AcrB [ 43 , 113 , 114 ], CusA [ 115 , 116 ], MtrD [ 117 , 118 ], CmeB [ 44 ], AdeB [ 119 ], HpnN [ 45 ] and MmpL3 [ 120 ]. We also used these protein purification protocols for in vitro substrate transport study via the CusA transporter [ 116 ] and in vitro functional dynamics measurement of the CmeB transporter [ 44 ], indicating that these purified membrane proteins are fully functional in vitro .…”

Efflux Pump Antibiotic Binding Site Mutations Are Associated with Azithromycin Nonsusceptibility in Clinical Neisseria gonorrhoeae Isolates

“…Lyu and Moseng et al used cryo-electron microscopy to characterize key residues involved in drug binding by mosaic-like MtrD efflux pump alleles in Neisseria gonorrhoeae ( 1 ). Isogenic experiments introducing key MtrD substitutions R714G and K823E increased macrolide MICs, leading the authors to predict that nonmosaic MtrD “gonococcal strains bearing both the mtrR promoter and amino acid changes at MtrD positions 714 or 823 could lead to clinically significant levels of Azi nonsusceptibility resistance.” We tested this hypothesis by analyzing a global meta-analysis collection of 4,852 N. gonorrhoeae genomes ( 2 ).…”