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

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 p...

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Published in:	Microbiology spectrum Vol. 10; no. 5; p. e0299022
Main Authors:	Lyu, Meinan, Ayala, Julio C, Chirakos, Isabella, Su, Chih-Chia, Shafer, William M, Yu, Edward W
Format:	Journal Article
Language:	English
Published:	United States American Society for Microbiology 26-10-2022
Subjects:	Amino Acids - metabolism Anti-Bacterial Agents - pharmacology Anti-Bacterial Agents - therapeutic use Anti-Infective Agents - pharmacology Bacterial Proteins - genetics Bacteriology Bile Acids and Salts - metabolism Cell Division Colistin - metabolism Coloring Agents - metabolism Coloring Agents - pharmacology Cryoelectron Microscopy Detergents - metabolism Detergents - pharmacology Drug Resistance, Multiple, Bacterial Humans membrane proteins MtrD multidrug efflux multidrug resistance Neisseria gonorrhoeae Novobiocin - metabolism Research Article secondary transporter mechanism multidrug resistance multidrug efflux Neisseria gonorrhoeae membrane proteins secondary transporter mechanism MtrD
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Summary:	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 ( ationic ntibiotic- ensitizing eptide). 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. RND efflux pumps can export numerous antimicrobials that enter Gram-negative bacteria, and their action can reduce the efficacy of antibiotics and provide decreased susceptibility to various host antimicrobials. Here, we identified a ationic ntibiotic- ensitizing eptide (CASP) that binds within the periplasmic cleft of an RND transporter protein (MtrD) produced by Neisseria gonorrhoeae. Surprisingly, CASP was able to render rod-shaped Gram-negative bacteria, but not gonococci, susceptible to an antibiotic that is a substrate for the gonococcal MtrCDE efflux pump. CASP (or its future derivatives) could be used as an adjuvant to treat infections for which RND efflux contributes to multidrug resistance.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 The authors declare no conflict of interest.
ISSN:	2165-0497 2165-0497
DOI:	10.1128/spectrum.02990-22