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

• Type of Material: Article
• Authors: Meinan Lyu, Case Western Reserve University; Mitchell A. Moseng, Case Western Reserve University; Jennifer L. Reimche, Emory University; Concerta L. Holley, Emory University; Vijaya Dhulipala, Emory University; Chih-Chia Su, Case Western Reserve University; William Shafer, Emory University; Edward W. Yu, Case Western Reserve University
• Language: English
• Date: 2020-05-01
• Publisher: American Society for Microbiology
• Publication Version: Final Published Version
• Copyright Statement: © 2020 Lyu et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1128/mBio.00996-20
• Title of Journal or Parent Work: mBio
• Volume: 11
• Issue: 3
• Grant/Funding Information: We gratefully acknowledge the support of Michael Rau and James Fitzpatrick who acquired the single-particle cryo-EM data at the Washington University Center for Cellular Imaging, which is funded, in part, by Washington University School of Medicine, the Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813), and the Foundation for Barnes-Jewish Hospital (3770).; This work was supported by NIH grants R01AI145069 (E.W.Y.) and R37AI21150 (W.M.S.) and funds from an Intergovernmental Personnel Act from the Centers for Disease Control and Prevention to J.L.R. and W.M.S. W.M.S. is the recipient of a Senior Research Career Scientist Award from the Biomedical Laboratory Research and Development Service of the U.S. Department of Veterans Affairs.; This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E.
• Keywords: Neisseria gonorrhoeae; Reveals; Crystal structure; Science & Technology; Mutations; Proteins; cryo-EM; Microbiology; System; Phenix; efflux pumps; structural biology; multidrug resistance; Antimicrobial resistance; Life Sciences & Biomedicine; MEXB
• Research Categories: Health Sciences, Immunology; Health Sciences, Pharmacology; Biology, Microbiology

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 Gramnegative 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 mosaiclike 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.