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Action and mechanism of the colistin resistance enzyme MCR-4.

ABSTRACT: Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (mcr) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of Shewanella frigidimarina via progressive evolution and forms a phylogenetically-distinct group from the well-studied MCR-1/2 family. Domain-swapping experiments further confirmed that MCR-1 and MCR-4 transmembrane and catalytic domains are not functionally-interchangeable. However, structural and functional analyses demonstrated that MCR-4 possesses a similar PE lipid substrate-recognizable cavity and exploits an almost-identical ping-pong catalysis mechanism. MCR-4 also can alleviate colistin-triggered accumulation of reactive oxygen species (ROS). Taken together, this finding constitutes a functional proof that MCR-4 proceeds in a distinct evolutionary path to fulfill a consistent molecular mechanism, resulting in phenotypic colistin resistance.

SUBMITTER: Zhang H

PROVIDER: S-EPMC6347640 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Action and mechanism of the colistin resistance enzyme MCR-4.

Zhang Huimin H Hou Mengyun M Xu Yongchang Y Srinivas Swaminath S Huang Man M Liu Lizhang L Feng Youjun Y

Communications biology 20190125

Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (<i>mcr</i>) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of <i>Shewanella frigidimarina</i> via progressive evolution and forms a phylogenetically ...[more]

PMID: 30701201

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Project description:Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem β-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2 Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1 Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4' position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens.IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.

Project description:Colistin (polymyxin E) and polymyxin B have been used as last-resort agents for treating infections caused by multidrug-resistant Gram-negative bacteria. However, their efficacy has been challenged by the emergence of the mobile colistin resistance gene mcr-1, which encodes a transmembrane phosphoethanolamine (PEA) transferase enzyme, MCR-1. The enzyme catalyzes the transfer of the cationic PEA moiety of phosphatidylethanolamine (PE) to lipid A, thereby neutralizing the negative charge of lipid A and blocking the binding of positively charged polymyxins. This study aims to facilitate understanding of the mechanism of the MCR-1 enzyme by investigating its active-site sequence requirements. For this purpose, 23 active-site residues of MCR-1 protein were randomized by constructing single-codon randomization libraries. The libraries were individually selected for supporting Escherichia coli cell growth in the presence of colistin or polymyxin B. Deep sequencing of the polymyxin-resistant clones revealed that wild-type residues predominates at 17 active-site residue positions, indicating these residues play critical roles in MCR-1 function. These residues include Zn2+-chelating residues as well as residues that may form a hydrogen bond network with the PEA moiety or make hydrophobic interactions with the acyl chains of PE. Any mutations at these residues significantly decrease polymyxin resistance levels and the PEA transferase activity of the MCR-1 enzyme. Therefore, deep sequencing of the randomization libraries of MCR-1 enzyme identifies active-site residues that are essential for its polymyxin resistance function. Thus, these residues may be utilized as targets to develop inhibitors to circumvent MCR-1-mediated polymyxin resistance. IMPORTANCE Polymyxin antibiotics are used as last-line antibiotics in treating infections caused by multidrug-resistant pathogens. However, widespread use of polymyxins has led to the emergence of resistance. Although multiple mechanisms for resistance exist, that due to mcr-1 is a particular concern, as it can be readily transferred among bacterial pathogens. The mcr-1 gene encodes a transmembrane phosphoethanolamine (PEA) transferase that modifies lipid A to block the binding of polymyxin antibiotics. We utilized random mutagenesis coupled with next-generation sequencing to determine the amino acid sequence requirements of 23 residues in and near the active site of MCR-1. We show that the enzyme has stringent sequence requirements, with 75% of the residues examined being essential for function. Coupled with the finding that these residues are largely conserved among PEA enzymes, the results suggest inhibitors that bind near these sites will broadly inhibit MCR-1 and other enzymes of this class.

Project description:Colistin resistance due to the mcr-type genes in Escherichia coli is well characterized. In order to study the resistance mechanism in mcr-negative colistin-resistant E. coli, strains were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. A total of 11 mcr-negative colistin-resistant isolates among 7,942 (0.1%) clinical E. coli isolates were identified between 2008 and 2018. Their prevalence was low and remained stable during the study period. Since 2012, ST131 and ST1193 clones with multiple drug-resistant phenotypes have emerged. All resistant strains displayed higher expression levels of the operons pmrHFIJKLM and pmrCAB than the control MG1655 strain. Although several amino acid substitutions were identified in PmrA or PmrB, only R81H in PmrA was associated with overexpression of pmrHFIJKLM and colistin resistance. The effect of substitution R81H in PmrA in colistin resistance was confirmed by complementation experiments. Although some strains harbored substitutions in PmrB, the identified mutations in pmrB did not contribute to colistin resistance. In conclusion, the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. IMPORTANCE The molecular epidemiology and resistance mechanisms of mcr-negative colistin-resistant E. coli are not well described. In this study, a total of 11 mcr-negative colistin-resistant E. coli isolates were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. We determined the resistance mechanism of non-mcr colistin-resistant strains using gene knockout and complementation experiments. We observed the occurrence of the global multiple-drug-resistant E. coli clones ST131 and ST1193 starting in 2012. Moreover, for the first time, we proved that the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. The study results helped to gain an insight into the diversity and complexity of chromosome-encoded colistin resistance in E. coli.

Dataset Information

Action and mechanism of the colistin resistance enzyme MCR-4.

Publications

Action and mechanism of the colistin resistance enzyme MCR-4.

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