Abstract
The emergence and worldwide dissemination of plasmid-mediated colistin resistance gene mcr-1 has attracted global attention. The MCR-1 enzyme mediated colistin resistance by catalyzing phosphoethanolamine (PEA) transfer onto bacterial lipid A. However, the interaction partners of MCR-1 located in membrane protein in E. coli are unknown. Co-immunoprecipitation (Co-IP) and Mass Spectrometry were performed to define the interacting proteins of MCR-1. A total of three different anti-MCR-1 monoclonal antibody (mAbs) were prepared and 3G4 mAb was selected as the bait protein by compared their suitability for Co-IP. We identified 53, 13, and 14 interacting proteins in E. coli BL21 (DE3) (pET28a-mcr-1), E. coli BL21 (DE3) (pET28a-mcr-1-200), and E. coli DH5α (pUC19-mcr-1), respectively. Six proteins, including the stress response proteins DnaK (chaperone protein) and SspB (stringent starvation protein B), the transcriptional regulation protein H-NS, and ribosomal proteins (RpsE, RpsJ, and RpsP) were identified in all these three strains. These MCR-1-interacting proteins were mainly involved in ribosome and RNA degradation, suggesting that MCR-1 influences the protein biosynthesis through the interaction with ribosomal protein. Multidrug efflux pump AcrA and TolC were important interacting membrane proteins of MCR-1 referred to drug efflux during the PEA modification of the bacterial cell membrane. Overall, we firstly identified the functional interactome profile of MCR-1 in E. coli and discovered that two-component AcrA-TolC multidrug efflux pump was involved in mcr-1-mediated colistin resistance.
Highlights
Mcr-1, as an important plasmid-borne colistin resistant gene, has attracted much attention in recent years for its threat in the clinical efficacy of the last-resort antibiotic when treating multidrug– resistant (MDR) Gram-negative bacterial infections (Liu et al, 2016; Shen et al, 2016; Poirel et al, 2017). mcr-1 encodes a member of the family of phosphoethanolamine (PEA) transferases that decorates the lipid A headgroups of lipopolysaccharide of the outer membrane of Gram-negative bacteria through the addition of PEA (Anandan et al, 2017; Li et al, 2018)
We identified a total of 53 interacting proteins linked with ribosomal proteins (RplJ, RplK, RplO, RplL, RplC, RpmC, RplF, RplD, RplI, RpsU, RpsE, RpsJ, RpsP, and RpsN), stress response proteins (DnaK and Stringent starvation protein B (SspB)), metabolism (IDH1, SdhB, AcnB, ATPF0B, and GatB) and drug efflux system (AcrA, Lpp, OmpA, Pal, and TolC) in E. coli BL21 (DE3) (Table 2)
Using a reliable Co-IP assay couple with mass spectrometry, we reported here the first functional interactome of MCR-1 and identified the key interacting proteins in colistinresistant E. coli strains
Summary
Mcr-1, as an important plasmid-borne colistin resistant gene, has attracted much attention in recent years for its threat in the clinical efficacy of the last-resort antibiotic when treating multidrug– resistant (MDR) Gram-negative bacterial infections (Liu et al, 2016; Shen et al, 2016; Poirel et al, 2017). mcr-1 encodes a member of the family of phosphoethanolamine (PEA) transferases that decorates the lipid A headgroups of lipopolysaccharide of the outer membrane of Gram-negative bacteria through the addition of PEA (Anandan et al, 2017; Li et al, 2018). Mcr-1 encodes a member of the family of phosphoethanolamine (PEA) transferases that decorates the lipid A headgroups of lipopolysaccharide of the outer membrane of Gram-negative bacteria through the addition of PEA (Anandan et al, 2017; Li et al, 2018). Functional Interactome Profile of MCR-1 gram-negative bacteria possess multiple members of this family of enzymes that are engaged in the decoration of lipid A or the conserved inner core of the lipopolysaccharide (Needham and Trent, 2013). As an important resistant protein, the functional interactome of membrane protein MCR-1 was poorly understood. Affinity purification based on co-immunoprecipitation (Co-IP) coupled to mass spectrometry has become an important method of identifying protein interactome (Pankow et al, 2016; Maccarrone et al, 2017). We define the interacting proteins of MCR-1 using Co-IP and mass spectrometry in E. coli and characterize the protein-protein interaction (PPI) network of MCR-1
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