Polymyxins, a family of cationic antimicrobial cyclic peptides, act as a last line of defense against severe infections by Gram-negative pathogens with carbapenem resistance. In addition to the intrinsic resistance to polymyxin E (colistin) conferred by Neisseria eptA, the plasmid-borne mobilized colistin resistance gene mcr-1 has been disseminated globally since the first discovery in Southern China, in late 2015. However, the molecular mechanisms for both intrinsic and transferable resistance to colistin remain largely unknown. Here, we aim to address this gap in the knowledge of these proteins. Structural and functional analyses of EptA and MCR-1 and -2 have defined a conserved 12-residue cavity that is required for the entry of the lipid substrate, phosphatidylethanolamine (PE). The in vitro and in vivo data together have allowed us to visualize the similarities in catalytic activity shared by EptA and MCR-1 and -2. The expression of either EptA or MCR-1 or -2 is shown to remodel the surface of enteric bacteria (e.g., Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, etc.), rendering them resistant to colistin. The parallels in the PE substrate-binding cavities among EptA, MCR-1, and MCR-2 provide a comprehensive understanding of both intrinsic and transferable colistin resistance. Domain swapping between EptA and MCR-1 and -2 reveals that the two domains (transmembrane [TM] region and phosphoethanolamine [PEA] transferase) are not functionally exchangeable. Taken together, the results represent a common mechanism for intrinsic and transferable PEA resistance to polymyxin, a last-resort antibiotic against multidrug-resistant pathogens.
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.
Polymyxins such as colistin are antibiotics used as a final line of defense in the management of infections with multidrug-resistant Gram-negative bacteria. Although natural resistance to polymyxins is rare, the discovery of a mobilized colistin resistance gene () in gut bacteria has raised significant concern. As an intramembrane enzyme, MCR-1 catalyzes the transfer of phosphoethanolamine (PEA) to the 1 (or 4')-phosphate group of the lipid A moiety of lipopolysaccharide, thereby conferring colistin resistance. However, the structural and biochemical mechanisms used by this integral membrane enzyme remain poorly understood. Here, we report the modeled structure of the full-length MCR-1 membrane protein. Together with molecular docking, our structural and functional dissection of the complex of MCR-1 with its phosphatidylethanolamine (PE) substrate suggested the presence of a 12 residue-containing cavity for substrate entry, which is critical for both enzymatic activity and its resultant phenotypic resistance to colistin. More importantly, two periplasm-facing helices (PH2 and PH2') of the trans-membrane domain were essential for MCR-1 activity. MALDI-TOF MS and thin-layer chromatography assays provide both and evidence that MCR-1 catalyzes the transfer of PEA from the PE donor substrate to its recipient substrate lipid A. Also, the chemical modification of lipid A species was detected in clinical species of bacteria carrying Our results provide mechanistic insights into transferable MCR-1 polymyxin resistance, raising the prospect of rational design of small molecules that reverse bacterial polymyxin resistance, as a last-resort clinical option to combat pathogens with carbapenem resistance.
BackgroundMobilized resistance to colistin is evolving rapidly and its global dissemination poses a severe threat to human health and safety. Transferable colistin resistance gene, mcr-3, first identified in Shandong, China, has already been found in several countries in multidrug-resistant human infections. Here we track the spread of mcr-3 within 13 provinces in China and provide a complete characterization of its evolution, structure and function.MethodsA total of 6497 non-duplicate samples were collected from thirteen provinces in China, from 2016 to 2017 and then screened for the presence of mcr-3 gene by PCR amplification. mcr-3-positive isolates were analyzed for antibiotic resistance and by southern blot hybridization, transfer analysis and plasmid typing. We then examined the molecular evolution of MCR-3 through phylogenetic analysis. Furthermore, we also characterized the structure and function of MCR-3 through circular dichroism analyses, inductively coupled plasma mass spectrometry (ICP-MS), liquid chromatography mass spectrometry (LC/MS), confocal microscopy and chemical rescue tests.Findings49 samples (49/6497 = 0.75%) were mcr-3 positive, comprising 40 samples (40/4144 = 0.97%) from 2017 and 9 samples (9/2353 = 0.38%) from 2016. Overall, mcr-3-positive isolates were distributed in animals and humans in 8 of the 13 provinces. Three mcr-3-positive IncP-type and one mcr-1-bearing IncHI2-like plasmids were identified and characterized. MCR-3 clusters with PEA transferases from Aeromonas and other bacteria and forms a phylogenetic entity that is distinct from the MCR-1/2/P(M) family, the largest group of transferable colistin resistance determinants. Despite that the two domains of MCR-3 not being exchangeable with their counterparts in MCR-1/2, structure-guided functional mapping of MCR-3 defines a conserved PE-lipid recognizing cavity prerequisite for its enzymatic catalysis and its resultant phenotypic resistance to colistin. We therefore propose that MCR-3 uses a possible “ping-pong” mechanism to transfer the moiety of PEA from its donor PE to the 1(or 4′)-phosphate of lipid A via an adduct of MCR-3-bound PEA. Additionally, the expression of MCR-3 in E. coli prevents the colistin-triggered formation of reactive oxygen species (ROS) and interferes bacterial growth and viability.InterpretationOur results provide an evolutionary, structural and functional definition of MCR-3 and its epidemiology in China, paving the way for smarter policies, better surveillance and effective treatments.
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