Biochemical Mechanisms For MCR Colistin Resistance

Polymyxin are a group of cationic antimicrobial peptides that are the last line of defense against pan-drug resistance pathogens in clinical settings.However,the emergence of plasmid mediated polymyxin resistant mcr gene in the world has become a potential threat to destroy the clinical use of polymyxin.Worse still,little is known about the mechanism of polymyxin resistance mediated by MCR gene family.In this study,we systematically analyzed the genetic evolutionary,enzymatic structure,cellular adaptability mechanism of MCR gene family.It will enrich the understanding of the mechanism of “acquired/transferable colistin resistance”.The main results are as follows:(1)Through the sequence alignment of MCR family proteins,phylogenetic tree and bioinformatics analysis,we found that MCR family genes can be roughly divided into four subfamily or four subclades of evolutionary tree.Among them,MCR-1/2 is belong to the subfamily I,MCR-3 is the II subfamily,MCR-4 and MCR-5 are the third and fourth subclade respectively.And the members of subfamily I and II are very numerous.The detailed analysis of phylogenetic tree showed that MCR-1/2 was highly homologous with the phosphoethanolamine transferase encoded by Moraxella chromosome,which may be the precursor gene of mcr-1/2.It was also found that the mcr-3 had a high similarity with the phosphoethanolamine transferase gene encoded by Aeromonas,and there was diversity of phosphoethanolamine transferase in the genus,suggesting that the genus was the natural host of the subfamily of mcr-3.It is implication that the precursor genes of subclades of MCR originate from different host reservoirs.(2)Agar dilution method was used to determine the level of colistin resistance in MCR gene family.It was found that Ept A and mcr-1/2 /3 showed distinct colistin resistance level in E.coli.In vivo,it was analyzed that there was a peak of substrate modification of PEA in lipid A of bacteria by MALDI-TOF /TOF,which proved that mcr-1/2/3 could play the catalytic activity in the physiological state of bacteria.In vitro,the purified MCR proteins still have the activity of catalytic substrate under experimental conditions which was conformed by TLC and LC/MS.This also suggests that MCR proteins catalyze two lipid substrates(PE and lipid A)through “ping-pong mechanism”.(3)Through homology modeling,it was found that MCR family proteins have two domains: transmembrane domain and catalytic domain,which are connected by a hinge region.By means of molecular docking and dynamics simulation,we found that the MCR has two pockets of binding substrates(PE and lipid A),which could recognize two substrates through structure conformational changes.And the key amino acid residues that interact with the substrates have been demonstrated by directed-site mutagenesis.(4)Using fluorescence confocal,we found that the expression of MCR gene could affect the cell state of bacteria.Overexpression of MCR protein showed that the growth of bacteria was obviously retarded.And a large number of dead cells were found in bacteria carrying mcr genes by fluorescent dye staining and confocal microscopy,which proved that there was fitness cost effect of MCR family.As the same time,it was observed that bacteria could produce massive of active oxygen radicals(ROS)under the treatment of colistin,but the presence of MCR genes could quench the production of ROS.This proved that MCR genes could not only produce colistin resistance by remodeling the structure of lipid A,but also weaken the bactericidal effect of colistin by quenching the ROS.