A New Variant of the Colistin Resistance Gene MCR-1 With Co-resistance to Β-lactam Antibiotics Reveals a Potential Novel Antimicrobial Peptide

Abstract

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to β-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to β-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

Introduction

Colistin/Polymyxin is a last-resort antibiotic against infections caused by highly drug-resistant bacteria, particularly carbapenem-resistant Enterobacterales [1]. However, the emergence of a novel plasmid-mediated colistin resistance gene named mcr-1 in 2016 threatened the clinical effectiveness of colistin. Since then, instances of mcr-1-positive Enterobacterales (MCRPE) have been detected from various sources (including livestock, humans, animal food products, and the environment) and have disseminated globally, spreading to >40 countries in 5 continents [2–4]. Colistin has been used as an additive in livestock feed to promote growth and prevent infection in China since the 1980s, and the correlation between the spread of mcr-1 and colistin use in animal husbandry has been observed [5]. To prevent the continued spread of plasmid-borne mcr-1, the Chinese government banned the use of colistin as animal feed additive in 2017. Such policy resulted in remarkable reductions in the production as well as sale of colistin sulfate premix [6] and a subsequent drastic decline in mcr-1 prevalence [7]. Nonetheless, a low prevalence of mcr-1 was still detected among inpatients, likely associated with the approval of colistin for clinical use in China [5,6,8,9]. Furthermore, increased co-existence of mcr-1 and carbapenemase genes after the clinical introduction of polymyxin was recently reported [10]. Hence, there is an urgent need to develop new strategies to eliminate the spread of mcr-1 and prolong the use of colistin/polymixin as the last-resort antibiotic against carbapenem-resistant bacteria that carry mcr-1.

Materials and methods

Ethics statement

With the approval from Ethics Committee of Zhongshan School of Medicine on Laboratory Animal Care (reference number: SYSU-IACUC-2022-B0031), Sun Yat-sen University, all the animal experiments were conducted based on the standard of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Bacterial strains and growth conditions

The E. coli strains used in this research were ATCC 25922 and K-12 derivatives, including BW25113 and DH5-α. BW25113 cells were utilized to assay the influence of MCR-1 or M6 expression upon bacterial drug sensitivity, viability, and membrane permeability, while DH5-α cells acted as a cloning host for plasmid construction. 

Results

MCR-1 variant M6 induces co-resistance towards β-lactam antibiotics
Given that MCR-1 disrupts OM integrity [31,32] facilitating entry of various antibiotics, we hypothesized that specific mutations in mcr-1 could abolish this phenotype. We tested this hypothesis using our previously established mcr-1 mutant library, which encompassed 171,769 mutation genotypes [33], covering 99.96% (4858/4860) possible single-nucleotide mutations of mcr-1. Screening was performed at antibiotic concentrations below and above the minimal inhibitory concentration (MIC, 0.8–2× MIC) to distinguish mutants with low- and high-level resistance (S1A Fig and S1 Table). By counting the colony-forming unit (CFU) values evaluated from plates containing aminoglycosides (streptomycin, SM), tetracyclines (tetracycline, TET), quinolones (nalidixic acid, NAL), or glycopeptides (vancomycin, VAN), we observed that some colonies from MCR-1 library could grow on plates containing penicillin (ampicillin, AMP), cephalosporin (ceftazidime, CAZ), or carbapenem (imipenem, IMP) (S1B Fig). Moreover, we confirmed that the MCR-1 library exhibited higher viability than the control strains after exposure to β-lactams (S1C Fig and S2 Table). Next, 50 isolates of the MCR-1 library growing on the plates containing 2 × MIC CAZ or AMP were selected, and the mcr-1 genotypes of the selected isolates were identified by Sanger sequencing (S3 Table). Subsequently, we conducted MIC assays to evaluate the susceptibility of the selected strains upon CAZ, AMP, and FOX. Several mutants displayed low-level resistance to AMP or CAZ (S3 Table). 

Discussion

In this study, we identified an MCR-1 mutant harboring 2 point mutations within the linker domain, causing significant disruptions in lipid A. Unlike MCR-1, the mutant protein confers phenotypic co-resistance to β-lactam antibiotics while inducing severe lipid A perturbations. This disorder eventually results in growth arrest, membrane permeabilization, and activation of LDTs pathway. Moreover, we have identified a lipid A binding pocket that is critical for colistin resistance and bacterial membrane integrity. The mutated pocket in M6 exhibited enhanced affinity towards lipid A, potentially underpinning the β-lactams co-resistance phenotype. However, the most striking outcome of our research is that antimicrobial peptides derived from MCR-1 protein itself provide a new strategy to combat drug resistance.

Acknowledgments

The plasmid encoding bacterial membrane voltage sensor Vibac2 was a generous gift from Prof. Bai Fan’s lab.

Citation: Liang L, Zhong L-L, Wang L, Zhou D, Li Y, Li J, et al. (2023) A new variant of the colistin resistance gene MCR-1 with co-resistance to β-lactam antibiotics reveals a potential novel antimicrobial peptide. PLoS Biol 21(12): e3002433. https://doi.org/10.1371/journal.pbio.3002433

Academic Editor: Csaba Pál, Biological Research Centre, HUNGARY

Received: April 3, 2023; Accepted: November 14, 2023; Published: December 13, 2023

Copyright: © 2023 Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files. The custom code used in this research for proteomics analysis, MCR proteins homology analysis and homology protein modelling were deposited in https://github.com/LiangLujie/MCR-1_new_variant and https://github.com/Wang-Lin-boop/AutoMD. The protein structure of MCR-1 and its lipid A binding is presented in S1 Supplementary PDB file.

Funding: This work was supported by the National Natural Science Foundation of China (grant number 81830103, 82061128001 and 82325033 to G-BT, grant number 82002173, 82272378 to SF), Natural Science Foundation of Guangdong Province (grant number 2017A030306012 to G-BT, grant number 2023A1515012392 to SF), Scientific and Technological Planning Project of Guangzhou City (grant number SL2022A04J01941 to SF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors declare no conflicts of interest.

Abbreviations: AMP, antimicrobial peptide; CFU, colony-forming unit; CRE, carbapenem-resistant Enterobacteriaceae; GO, Gene Ontology; IM, inner membrane; LB, Luria–Bertani; LPS, lipopolysaccharide; MCRPE, mcr-1-positive Enterobacterales; NPN, n-phenyl-1-napthylamine; OM, outer membrane; PE, phosphatidylethanolamine; PEA, phosphoethanolamine; PI, propidium iodide; PG, peptidoglycan; PPI, protein–protein interaction; SEM, scanning electron microscopy; sfGFP, super-folded green fluorescent protein; TEM, transmission electron microscopy

https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002433#abstract0