The gene encoding mcr-1, was amplified by polymerase chain reaction (PCR) from E. coli strain SHP45 genomic DNA using primers pET28a-mcr-1-F and pET28a-mcr-1-R and cloned into the plasmid pET28a to create a high copy expression vector with a N terminal hexa-histidine tag (pET28a-mcr-1) (Supplementary Table 1). Then the constructed plasmid pET28a-mcr-1 was chemically transferred into E. coli BL21 (DE3) strains to express the full length MCR-1. For the expression of the catalytic domain of MCR-1, the constructed plasmid pET28a-mcr-1-200 using primers pET28a-mcr-1-200-F and pET28a-mcr-1-200-R was transformed into E. coli BL21 (DE3) strains (Supplementary Table 1). Transformants were selected by the inclusion of kanamycin (50 μg/ml) on Luria Broth (LB) agar plates. The construction of E. coli cloning strains [E. coli DH5α (pUC19) and DH5α (pUC19-mcr-1)] was referred to our previous article (Li et al., 2019). The bacterial strains and plasmids used in this study were listed in Table 1. A starter culture was prepared by inoculating a single colony into 2 ml LB medium and grown at 37°C overnight. A sufficient volume of the starter culture was used to inoculate 200 ml LB medium and grown at 37°C in a shaking incubator set at 200 rpm until the OD₆₀₀ reached 0.5–0.6. Protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and the culture was continued at 18°C, 160 rpm for a further 14 h. The cells were harvested by centrifuging at 9,000 × g for 20 min at 4°C.

The harvested cells were re-suspended in 50 mM sodium phosphate buffer pH8.0 containing 300 mM NaCl, 10 mM imidazole at 4°C. Between 5 and 10 ml was used for each gram of cell pellet and cell lysis was performed using VCX105 ultrasonic instrument (Sonics & Materials, Inc., Newtown, CT, United States). The lysate was centrifuged at 12,000 × g for 30 min at 4°C to remove the non-lysed cells. The protein was purified from the supernatant using Ni-NTA agarose beads (Qiagen, Hilden, Germany). Briefly, the supernatant was incubated with pre-equilibrated Ni-NTA agarose beads (1 ml per pellet from a 200 ml culture) for 1 h. The beads were then loaded on a column and washed with 50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole (pH 8.0). Protein was eluted with four column volumes of 50 mM NaH₂PO₄, 300 mM NaCl, and 150 mM imidazole (pH8.0). Imidazole was removed from the eluted protein by exchanging buffer to 50 mM NaH₂PO₄, 300 mM NaCl, and 1 mM tris (2-carboxyethyl) phosphine (TCEP) (pH8.0) using a PD-10 desalting column (GE Healthcare).

The purified MCR-1 catalytic protein was used for immunization and custom production of the affinity-purified mouse mAb was used for subsequent experiments. All animal treatments were in accordance with Chinese laws and guidelines that were approved by the Animal Ethics Committee of China Agricultural University. Six eight-week old female BALB/c mice were immunized with the immunogen. The mice were immunized three times with an interval of 3 weeks between immunizations. The collected ascites were further purified to obtain the anti-MCR-1 mAb using AKTA Pure 150 system (GE Healthcare, Buckinghamshire, United Kingdom). Specificity of the antibody was determined by SDS-PAGE and Western blot.

To identify the interactome profile associated with MCR-1, three E. coli clone strains carrying mcr-1 gene [DH5α (pUC19-mcr-1), BL21 (DE3) (pET28a-mcr-1), and BL21 (DE3) (pET28a-mcr-1-200)] and two empty vector strains [DH5α (pUC19) and BL21 (DE3) (pET28a)] were included to pull down MCR-1 and associated proteins using Co-IP assays with anti-MCR-1 mAb 3G4 as the bait. Anti-IgG and empty vector pull-downs were set as negative controls. The Co-IP assays of membrane protein MCR-1 was performed as previously described (Pankow et al., 2016). Briefly, the cells of two cloning strains [(DH5α (pUC19), DH5α (pUC19-mcr-1)] and three expressing strains [BL21 (DE3) (pET28a), BL21(DE3) (pET28a-mcr-1), and BL21(DE3) (pET28a-mcr-1-200)] were lysed by 50 mM Tris–HCl (pH7.5), 300 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% n-dodecyl β-D-maltoside (DDM). 1 mg of solubilized proteins were incubated overnight with 250 μg of anti-MCR-1 mAb in a reaction volume of 100 μl at 4°C with gentle mixing. 50 μl of pre-equilibrated G-Sepharose (GE Healthcare, Uppsala, Sweden) was added and incubated for 4 h at 4°C with gentle agitation. After centrifugation, samples were washed 5 times with solubilization buffer and co-precipitates were eluted by incubation with 0.2 M glycine (pH2.3) and 0.5% Igepal CA-630 (Sigma-Aldrich, St. Louis, MO, United States). Co-precipitates were separated by SDS-PAGE, and visualized with silver staining. The MCR-1 binding proteins were identified using nano LC-MS/MS analysis. Three biological repeats were conducted in the Co-IP assay.

The co-precipitates were analyzed on a Q-exactive mass spectrometer equipped with a Dionex Ultimate 3000 Nano (Thermo Fisher Scientific). Peptides were trapped on a Acclaim PePmap 100 (75 μm × 2 cm, nanoviper, C₁₈, 3 μm, Thermo Fisher Scientific) and separated on a Venusil XBP C₁₈ (2.1 × 150 mm, 5 μm, Agela Technologies) using a gradient formed between solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The gradient started at 5% solvent B and the concentration of solvent B was increased to 80% within 60 min. Database search was performed using the MASCOT software against the E. coli K12 database.

The surface plasmon resonance (SPR) interaction analysis was performed using the Reichert 4SPR instrument (Reichert Life Sciences). The MCR-1 and MCR-1-200 protein in PBS buffer were directly immobilized on a Planar Mixed SAM biosensor chip (Reichert Life Sciences), respectively. SspB was sequentially diluted in running buffer PBST (pH7.4, containing 0.05% Tween-20) and injected to be trapped on the chip through the immobilized MCR-1. Evaluation and calculation of the binding parameters were carried out according to the formula: K_D = K_a/K_d (K_a = association rate constant and K_d = dissociation rate constant).

A protein-protein interaction (PPI) network was constructed from the STRING database v11.0 (Szklarczyk et al., 2017) using the set of MCR-1 interacting proteins identified here. The interaction score was set as medium confidence (more than 0.400), and gene ontology (GO) enrichment was calculated by Blast2GO software. Metabolic pathway of MCR-1 interacting proteins was analyzed using KEGG software¹ to facilitate the related biological interpretation.

Recombinant plasmid pET28a-mcr-1 was induced by IPTG to express a 6 × His-tagged recombinant protein at the N-terminus, consisting of 541 amino acids. The molecular weight of MCR-1 full-length protein after fusion expression was about 66 KDa. The catalytic domain of MCR-1 was induced and expressed by recombinant plasmid pET28a-mcr-1-200. The catalytic domain of MCR-1 consisted of 341 amino acids and its molecular weight was about 46 KDa. IgG positive hybridoma cells belonging to three isotypes-IgG1 (3G4, 6G4, and 8H11) were obtained. The anti-MCR-1 mAbs were purified from ascites fluid by protein G resin affinity chromatography. The cell lysate of the constructed E. coli DH5α (pUC19-mcr-1) strains was used for anti-MCR-1 mAb validation through regular Western blot method. His6-MCR-1 bands were detected by anti-His6 mAb (Figure 1A) and MCR-1 were successfully recognized by newly prepared anti-MCR-1 mAbs (Figure 1B). Then we compared the suitability of an antibody for Co-IP in a small-scale experiment followed by western blotting. We found that the anti-MCR-1 mAb 3G4 had no cross-linking efficiency and optimal performance in pulling down the target protein (Figure 1C).

In an effort to better understand the mechanistic role of MCR-1, Co-IP, and mass spectrometry were used to identify the interacting proteins of MCR-1 in E. coli. Corresponding strong band at the expected size of MCR-1 and MCR-1-200 and a large number of other immunoprecipitated protein bands were shown using SDS-PAGE and silver stained (Figure 1D). 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 SspB), metabolism (IDH1, SdhB, AcnB, ATPF0B, and GatB) and drug efflux system (AcrA, Lpp, OmpA, Pal, and TolC) in E. coli BL21 (DE3) (pET28a-mcr-1) (Table 2). In addition, we identified a total of 14 interacting proteins in E. coli DH5α (pUC19-mcr-1) and these interacting proteins mainly included ribosomal proteins (RpsE, RplK, RpsJ, RpsP, and RplK), DNA-binding proteins (H-NS, GntR, and EF-Tu) and stress response proteins (DnaK, GroL, and SspB) (Supplementary Table 2). A total of 13 interacting proteins including ribosomal proteins (RpsJ, RpsE, and RpsP), DNA-binding proteins (H-NS, HupA, HupB, and HofN) and stress response proteins (DnaK and SspB) were identified in the soluble domain expression vector E. coli BL21 (DE3) (pET28a-mcr-1-200) (Supplementary Table 3). The number of MCR-1-interacting proteins identified in full-length protein-expressing strains BL21 (DE3) (pET28a-mcr-1) was significantly higher than that in catalytic domain-expressing strains BL21 (DE3) (pET28a-mcr-1-200). Next, we analyzed all of the MCR-1-interacting proteins using InteractiVenn software. Of these proteins, six proteins (DnaK, SspB, H-NS, RpsE, RpsJ, and RpsP) were identified in all the three strains (Figure 1E).

To elucidate the functional relationships between the MCR-1 interacting proteins, we extracted a PPI network over these proteins from the STRING interaction database. We identified several functional proteins associated with ribosome, DNA replication, and hyperosmotic shock in E. coli DH5α (pUC19-mcr-1) (Figure 2A). The interacting proteins of MCR-1 in E. coli BL21 (DE3) (pET28a-mcr-1) were mainly involved in ribosome, drug efflux system, DNA binding, and DNA replication (Figure 2B). And the MCR-1-interacting proteins in E. coli BL21 (DE3) (pET28a-mcr-1-200) were mainly related with ribosome and DNA binding (Figure 2C). KEGG pathway enrichment analysis of the MCR-1-interacting proteins in E. coli DH5α (pUC19-mcr-1) found that these proteins were mainly involved in ribosome and RNA degradation (Figure 2D). And the KEGG pathway of the MCR-1-interacting proteins identified in E. coli BL21 (DE3) (pET28a-mcr-1) were referred to ribosome, RNA degradation, and cationic antimicrobial peptide (CAMP) resistance (Supplementary Figure 1A). Interestingly, multidrug efflux pump AcrA and TolC was identified important interacting partners of MCR-1 (Table 2). Outer membrane lipoprotein SlyB contributes to membrane integrity. YidC is required for the insertion and/or proper folding of integral membrane proteins into the membrane. The discovery of these interacting proteins demonstrated that MCR-1 might affect the expression and regulation of membrane proteins to cause drug efflux during the PEA modification of bacterial cell membrane lipid A.

To identify putative functional processes associated with MCR-1-interacting proteins, we performed GO analysis. In E. coli DH5α (pUC19-mcr-1), the top-ranked categories of Biological Process analysis were cellular macromolecule biosynthetic process, cellular component biogenesis, protein-containing complex assembly (Figure 2E), suggesting that MCR-1 is related to protein biosynthetic process. In addition, Molecular Function of the MCR-1 interacting partners referred to organic cyclic compound binding, heterocyclic compound binding and RNA binding (Figure 2E). It indicates that MCR-1 may be involved in nucleic acid binding. Cellular Component analysis showed that these proteins were related to protein-containing complex, cytosolic part, and ribosome (Figure 2E), which implies that MCR-1 is likely to participate in protein biosynthesis. GO analysis of MCR-1-interactiong proteins in E. coli BL21 (DE3) (pET28a-mcr-1) were shown in Supplementary Figures 1B–D.

To confirm the reliability of the developed Co-IP method and the effects of the identified MCR-1-interacting proteins, we investigated the binding ability of the target protein MCR-1, MCR-1-200, and SspB on the sensor chip. The SPR responses were obtained when the protein solution (0.156–5 μM, twofold dilution) was injected onto the sensor chip. The K_D values for the association between the MCR-1 full length protein and the SspB were 1.40 × 10^–4 and 5.13 × 10^–6 M for catalytic domain protein MCR-1-200, respectively (Figures 3A,B). These results demonstrated that MCR-1 was directly interacted with SspB in colistin-resistant E. coli strains.