Dissemination and Mechanism for the MCR-1 Colistin Resistance

Abstract

Polymyxins are the last line of defense against lethal infections caused by multidrug resistant Gram-negative pathogens. Very recently, the use of polymyxins has been greatly challenged by the emergence of the plasmid-borne mobile colistin resistance gene (mcr-1). However, the mechanistic aspects of the MCR-1 colistin resistance are still poorly understood. Here we report the comparative genomics of two new mcr-1-harbouring plasmids isolated from the human gut microbiota, highlighting the diversity in plasmid transfer of the mcr-1 gene. Further genetic dissection delineated that both the trans-membrane region and a substrate-binding motif are required for the MCR-1-mediated colistin resistance. The soluble form of the membrane protein MCR-1 was successfully prepared and verified. Phylogenetic analyses revealed that MCR-1 is highly homologous to its counterpart PEA lipid A transferase in Paenibacili, a known producer of polymyxins. The fact that the plasmid-borne MCR-1 is placed in a subclade neighboring the chromosome-encoded colistin-resistant Neisseria LptA (EptA) potentially implies parallel evolutionary paths for the two genes. In conclusion, our finding provids a first glimpse of mechanism for the MCR-1-mediated colistin resistance.

Fig 1. Working model proposed for MCR-1-catalyzed reaction in E. coli.

A. The chemical mechanism for MCR-1-mediated colistin resistance MCR-1 catalyzes the reaction of PPEA-4’-lipid A generation from lipid A plus phosphatidylethanolamine in which diacylglycerol is also one end product. In light of its similarity to the Neisseria LptA [2], the MCR-1-mediated reaction was given, in which the chemical structures of molecules were generated using the software ChemDraw. B. Silver staining analyses for the isolated E. coli lipopolysaccharide (LPS) containing lipid A (LPS-Lipid A). The bacterial LPS were isolated as described by Wanty et al. [2] with appropriate modifications.

Fig 2. Scheme for the two mcr-1-harbouring plasmids pE15004 and pE15017.

A. Genomic map of the mcr-1-containing IncX4-type plasmid pE15004 from the human gut microbiota. B. Genomic map of the MCR-1 and ESBL-coproducing IncI2-type plasmid pE15017 from the human gut microbiota. Circles from inside to outside indicate the GC screw, GC content and the open-reading frames in different DNA strands. The plasmid sequences were annotated by RAST, and the maps were generated using Circos program.

Fig 3. Genetic features of the two mcr-1-positive plasmids (pE15004 and pE15017).

A. Schematic representation of the ESBL and MCR-1-coproducing plasmid pE15017. Arrows denote the genes with specific transcriptional direction. The mcr-1 gene is in red, whereas the ESBL-encoding gene bla _CTX-M-55 is indicated in blue. The cassette of ISEcp1-bla _CTX-M-55-orf477 is highlighted in blue background, and the mcr-1-containing mobile element is under-scored in yellow background. The fragment “6” (earlier denoted as tnpA*) means the inter-space region adjacent to the 3’-end of the tnpA. A set of PCR primers (S2 Table) were designed to further confirm the presence of the two cassettes of “ISEcp1-bla _CTX-M-55-orf477” and “tnpA*-mcr-1-hp”, as well as their neighboring loci and/or virulence factors like virD4 [10]. B. PCR assays for the cassette of ISEcp1-bla _CTX-M-55-orf477 and its neighboring loci on the plasmid pE15017. C. PCR-based detection of the mcr-1 gene and other six loci. Designations: nikB, a relaxase for transposon; pilP, a Type IV pilus biogenesis protein; virD4-virB4, two genes encoding two components type IV secretion system; tnpA, a transposase-encoding gene; and hp, a hypothetical protein. M refers to Trans 2K Plus II DNA Ladder (TRANSGEN BIOTECH, Beijing, China), and kb denotes kilo-base pair. D. Scheme for genetic organization of the ISEcp1-bla _CTX-M-55-orf477 operon from pE15017, pHN122-1 and pA31-12. The ISEcp1-bla _CTX-M-55-orf477 cassettes on the two plasmids (pE15017 and pA31-12) are identical and only 4bp shorter than that of pHN122-1. E. Schematic representation of the mcr-1-containing mobile elements from the different plasmids. The insertion sequence ISApl1 occurs in both pHNSHP45 and pA31-12 is absent in the plasmid pE15017, which is validated by PCR detection coupled with Sanger sequencing. Similarly, no insertion sequence is found in front of the mcr-1 gene in the two incX4-type plasmids pE15004 and pMCR1-IncX4. Unlike the DR (TTTTC) for the ISEcp1-bla _CTX-M-55-orf477 (in Panel D), the DR sequences for the mcr-1-hp with/without ISApl1 (in Panel E) are divergent (GA in pHNSHP45, GA/GAA in the two IncI2 plasmids (pE15017 plus pA31-12), and CGG in the two IncX4 plasmids (pE15004 & pMCR1-IncX4)). Abbreviations: DR, Direct Repeats; IRL, l Inverted Repeats at Left; IRR, Inverted Repeats at Right. IRR2 is indicated with red rectangle. The sequences of the repeats are listed in the box on the right hand. X denotes the deleted nucleotide.

Fig 4. Structure-guided determination of five important residues for MCR-1 mediated colistin resistance.

A. The modeled ribbon structure for PEA-lipid A transferase domain of the membrane-bound MCR-1 protein. The ribbon structure was given via PyMol software. The key residues proposed by structural docking is indicated with red rectangle. B. The enlarged view of the five crucial residues for PEA-lipid A transferase activity of the MCR-1 protein. The five important residues include E246, T285, H395, D465 and H466, respectively. C. Structural-guided functional determination of the five residues (E246, T285, H395, D465 and H466) essential for MCR-1-mediated colistin resistance. A representative result of three independent experiments is given. Note: panel C is generated using the photograph combined with two plates because that plate size is limited and not allowed us to spot all the samples in a same plate.

Fig 5. Phylogeny of MCR-1.

The method of maximum likelihood tree is applied here. The scale bar corresponds to a 100% difference in compared residues, on average, per branch length. The members in this phylogenetic tree can be grouped into two clades (one is annotated as Phosphoethanolamine transferases [PEA transferase], and the other denotes Sulfatases [marked in pink]. Of note, the group of PEA transferase can be divided into two sub-clades: Sub-clade I (marked in blue) with MCR-1 and Sub-clade II (highlighted in green) with Neisseria gonorrhoeae LptA.