Identification of Functional Interactome of Colistin Resistance Protein MCR-1 in Escherichia coli

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.

INTRODUCTION 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 multidrugresistant (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 belongs to the alkaline phosphatase superfamily/sulphatase with five transmembrane segments. Many MDR 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). The proper function of an organism is orchestrated by highly complex protein networks and the interactome is crucial for understanding structural and functional organization in time and space (Sanchez et al., 1999). Deciphering the interactome profile of MCR-1 may help us in decoding its exact physiological function and various related biological process.
At present, the dissemination and prevalence of mcr-1 has been widely reported in human, foods, animals, and environment Ye et al., 2016;Wang et al., 2017). 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). Discovering interaction protein is still challenging, however, because a comprehensive interactome analysis requires high yields of the bait protein for robust co-purification of its interactors. In this study, we express and purify the catalytic domain and full-length protein of MCR-1, and prepare three different anti-MCR-1 monoclonal antibody (mAbs). 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.

Expression and Purification of MCR-1
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 OD 600 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 NaH 2 PO 4 , 300 mM NaCl, and 20 mM imidazole (pH 8.0). Protein was eluted with four column volumes of 50 mM NaH 2 PO 4 , 300 mM NaCl, and 150 mM imidazole (pH8.0). Imidazole was removed from the eluted protein by exchanging buffer to 50 mM NaH 2 PO 4 , 300 mM NaCl, and 1 mM tris (2-carboxyethyl) phosphine (TCEP) (pH8.0) using a PD-10 desalting column (GE Healthcare).

Preparation of MCR-1 mAb
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.

Nano LC-MS/MS and Data Analysis
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, C 18 , 3 µm, Thermo Fisher Scientific) and separated on a Venusil XBP C 18 (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.

Surface Plasmon Resonance Interaction Analysis of MCR-1 and SspB Protein
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).

Bioinformatics Analysis
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 software 1 to facilitate the related biological interpretation.

Protein Expression and mAb Preparation of MCR-1
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).
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 (pUC19mcr-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 Frontiers in Microbiology | www.frontiersin.org  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) (pET28amcr-1) were shown in Supplementary Figures 1B-D.

Interaction Analysis of SspB With MCR-1
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.

DISCUSSION
With the rapid spread of the mcr-1, its public health problems and human health threats need to be solved urgently. The identification of functional interaction partners is fundamental to understanding the role of proteins and the mechanism of action in vivo. This is particularly challenging because bacteria will continue to produce adaptive changes under the pressure of antibiotics. 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.
Our data indicate that MCR-1 interacts with multiple types of proteins. Among the interacting proteins identified by mass spectrometry, the most enriched groups of proteins associated with MCR-1 were the components of the ribosome and cellular stress response ( Figure 2D). We speculate that MCR-1 interact with ribosome-associated proteins to affect protein biosynthesis in conferring resistance by modifying the colistin target, catalyzing transfer of phosphoethanolamine (PEA) onto the glucosamine saccharide of lipid A in the bacterial outer membrane. Our previous research indicated that the expression of some ribosomal proteins was disturbed in the construct E. coli strains carrying mcr-1 and the bacteria could enhance protein synthesis in order to adapt to drug selection pressure (Li et al., 2019). Evaluating the likelihood and functional impact of the ribosome-associated interacting partners of MCR-1 will be an interesting follow up of this study.
As known, AcrAB-TolC multidrug resistance pump provides the Gram-negative bacteria with the necessary means to adapt drug pressure (Nikaido and Takatsuka, 2009). AcrA is the adapter component that associates the inner membrane pump with the TolC outer membrane channel (Fralick, 1996;Symmons et al., 2009). Our previous research found that MCR-1 not only caused the PEA modification of bacterial cell membrane lipid A, but also affected the efflux of polymyxin through disturbing the expression of efflux pump proteins involved in CAMP resistance pathway (Li et al., 2019). This study further confirmed that AcrA and TolC were important interacting membrane proteins of MCR-1 referred to drug efflux process. This finding indicated that MCR-1 might cause bacterial cell membrane to undergo the PEA molecule modification, which might also cause efflux pumps to participate in this biological process. The identification of the interacting partners SlyB and YidC showed that the importance of membrane protein integrity in mcr-1-mediated colistin resistance in E. coli. Lpp was a protein required for maintaining structural and functional integrity of bacterial cell envelope (Stenberg et al., 2005). Lpp was an integral component of cell outer membrane and seemed to interact with TolB, Pal, and TonB. We also found that H-NS interacted with DnaK, Lpp, HupA, and HupB in our study ( Figure 2B). The regulatory protein H-NS controlled the lipid A palmitoylation mediated by the PagP enzyme (Chalabaev et al., 2014). Future work is needed to uncover the role of the promiscuous interaction of MCR-1 with two-component efflux pump AcrA-TolC and the contribution of other membrane proteins of the MCR-1 interactome during colistin resistance.
The stringent response is a general bacterial stress response that allows bacteria to adapt and survive adverse conditions. The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (e.g., DnaK) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. Here, we found DnaK was a chaperone protein that interacted with MCR-1 ( Figure 1E and Table 2). Chaperone protein DnaK was a multifunctional chaperone of highly conserved HSP70 family which assisted in protein folding, disaggregation, and remodeling of protein complexes (Calloni et al., 2012;Zahn et al., 2013). DnaK functions as a central hub to interact with a large number of proteins to regulate ribosomal biogenesis in E. coli . DnaK has been proven to play an important role in the stress resistance of microorganism and may associate with the fitness cost reduction for mcr-1-carrying plasmids (Genevaux et al., 2007;Ma et al., 2018). We elucidate that DnaK might assist in the regulation of ribosomal biogenesis and affect the lipopolysaccharide modification. SspB, another protein that has been shown to interact with MCR-1 using Co-IP and SPR (Figure 3). Stringent starvation protein B (SspB) enhanced recognition of SsrA-tagged proteins by the ClpX-ClpP protease and regulated the protein expression during exponential and stationary-phase growth (Levchenko et al., 2000). H-NS was a global DNA-binding transcriptional dual regulator and implicated in transcriptional repression. RpsE, RpsJ, and RpsP are components of 30S ribosomal subunit and play an important role in ribosome biosynthesis. RpsE linked to the functional center of the 30S ribosomal subunit and was implicated in translational accuracy (Wimberly et al., 2000;Kirthi et al., 2006). RpsJ is involved in the regulation of ribosomal RNA biosynthesis by transcriptional antitermination (Luo et al., 2008;Weisberg, 2008). RpsP is essential for the viability of E. coli and plays an important role in the assembly of the 30S ribosomal subunits (Persson et al., 1995). So we predicted that MCR-1 interacted with H-NS to inhibit the DNA transcription and linked with ribosome proteins (RpsE, RpsJ, RpsP, etc.). Importantly, the identification and validation of MCR-1 interaction partner SspB with proven relevance demonstrate the power of our new Co-IP assay and provide reliable protein targets to advance our understanding of the mcr-1-mediated colistin resistance.
In conclusion, we define the functional interactome profile of colistin resistant protein MCR-1 in E. coli strains using Co-IP and mass spectrometry. Our study has uncovered a conceivable mechanism that MCR-1 influences the protein biosynthesis through the interaction with ribosomal protein. Multidrug efflux pump AcrA and TolC involved in the cationic antimicrobial peptide (CAMP) resistance pathway were identified as important interacting partners of MCR-1. Our data illustrates the interacting network of MCR-1 in colistin resistance and can provide valuable information to accurately understand its function and the mechanism of action at a deeper level.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by Chinese laws and guidelines that were approved by the Animal Ethics Committee of China Agricultural University.

AUTHOR CONTRIBUTIONS
BS, JS, YW, and XX conceived and designed the experiments. HL, YW, and QC performed the experiments. HL, YW, QC, and XX analyzed the data. HL, YW, and BS wrote the manuscript. All authors contributed to the article and approved the submitted version.