Structural Basis of Peptide-Based Antimicrobial Inhibition of a Resistance-Nodulation-Cell Division Multidrug Efflux Pump

• Authors: Meinan Lyu, Case Western Reserve University; Julio C Ayala, Emory University; Isabella Chirakos, Case Western Reserve University; Chih-Chia Su, Case Western Reserve University; William Shafer, Emory University; Edward W Yu, Case Western Reserve University
• Journal Title: MICROBIOLOGY SPECTRUM
• Volume: Volume 10, Number 5
• Publisher: AMER SOC MICROBIOLOGY | 2022-09-19, Pages e0299022-e0299022
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1128/spectrum.02990-22
• Subjects: Biology, Cell
• Keywords: Science & Technology; Life Sciences & Biomedicine; Microbiology; MtrD; Neisseria gonorrhoeae; membrane proteins; multidrug efflux; multidrug resistance; secondary transporter mechanism; NEISSERIA-GONORRHOEAE; ESCHERICHIA-COLI; CRYO-EM; PROTEIN; SYSTEM; TRANSPORTER; IDENTIFICATION; SUSCEPTIBILITY; ANTIBIOFILM; LIBRARIES

Abstract

Bacterial efflux pumps in the resistance-nodulation-cell division (RND) family of Gram-negative bacteria contribute significantly to the development of antimicrobial resistance by many pathogens. In this study, we selected the MtrD transporter protein of Neisseria gonorrhoeae as it is the sole RND pump possessed by this strictly human pathogen and can export multiple antimicrobials, including antibiotics, bile salts, detergents, dyes, and antimicrobial peptides. Using knowledge from our previously published structures of MtrD in the presence or absence of bound antibiotics as a model and the known ability of MtrCDE to export cationic antimicrobial peptides, we hypothesized that cationic peptides could be accommodated within MtrD binding sites. Furthermore, we thought that MtrD-bound peptides lacking antibacterial action could sensitize bacteria to an antibiotic normally exported by the MtrCDE efflux pump or other similar RND-type pumps possessed by different Gram-negative bacteria. We now report the identification of a novel nonantimicrobial cyclic cationic antimicrobial peptide, which we termed CASP (cationic antibiotic-sensitizing peptide). By single-particle cryo-electron microscopy, we found that CASP binds within the periplasmic cleft region of MtrD using overlapping and distinct amino acid contact sites that interact with another cyclic peptide (colistin) or a linear human cationic antimicrobial peptide derived from human LL-37. While CASP could not sensitize Neisseria gonorrhoeae to an antibiotic (novobiocin) that is a substrate for RND pumps, it could do so against multiple Gram-negative, rod-shaped bacteria. We propose that CASP (or future derivatives) could serve as an adjuvant for the antibiotic treatment of certain Gram-negative infections previously thwarted by RND transporters.