A Single Residue within the MCR-1 Protein Confers Anticipatory Resilience

Abstract

The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz., the two-component signal transduction system CpxRA (conjugative pilus expression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers.

Keywords: E. coli; MCR-1; envelope stress response (ESR); lipid A; mass spectrometry; pH-enhanced resistance; plasmid elimination.

FIG 1

Specific cleavage of MCR-1 by DegP. (A) Immunoblot of MCR-1 in periplasmic fractions of clinical E. coli isolates V163, NRZ14408, E. coli 023, E. coli 053, and E. coli 032 carrying the mcr-1 gene. Full-length (55 kDa) and cleaved (38 kDa) forms of the protein were detected in all isolates regardless of MICs of colistin. (B) Purification of MCR-1 and detection of a cleaved 38 kDa derivative as observed by Coomassie blue staining following SDS-PAGE. Edman sequencing identified the 38 kDa polypeptide as a fragment of MCR-1 following its cleavage at aa 198. Lane 1, His-tagged purified MCR-1; lanes 2 and 3, MCR-1; lanes 4 and 5, MCR-1_199–541 following size exclusion chromatography. The full-length protein obtained from this experiment was used for the cleavage studies whose results are presented in panel D and Fig. 2F. (C) Location of the DegP cleavage site within the PBD connecting the N- and C-terminal domains of MCR-1. The crystal structure of MCR-1 was modeled using RoseTTAFold. The different regions of the proteins are labeled and depicted in different colors. (D) Coomassie-stained SDS-PAGE following incubation of purified MCR-1 with DegP. Lane 1, MW ladder; lane 2, MCR-1; lane 3, DegP; lane 4, DegP and MCR-1 coincubated. DegP* is a self-cleavage product of DegP. (E) Amino acid sequence of the PBD (aa 181 to 237) with the DegP cleavage site at aa 198 (in pink). (F) Comparison of the PBD of phylogenomically distant alleles of MCR-1 (see also Fig. S6).

FIG 2

Mutations at the cleavage site have different consequences for colistin resistance activity. (A) qRT-PCR analysis of E. coli DH10β expressing MCR-1_WT, MCR-1_P198A, MCR-1_P198Y, or MCR-1_H478A. Data are means and standard errors of the means (SEM) from three independent experiments (ns, not significant). (B) Immunoblots of periplasmic fractions isolated from E. coli DH10β expressing MCR-1 and MCR-1 mutants developed with anti-MCR-1 and anti-OmpA Abs. (C to E) Charge-deconvoluted MS spectra of lipid A preparations for E. coli DH10β strains expressing either MCR-1_WT, MCR-1_P198A, or MCR-1_P198Y performed in the negative-ion mode and recorded in an m/z window of 400 to 2,500. The m/z region shown includes penta- to hepta-acylated lipid A species. The typical biphosphorylated, hexa-acylated E. coli lipid A has a calculated mass (m/z) of 1,797.219 Da, and the single PEA-modified form has an m/z of 1,920.228 Da. Comparable levels of PEA modification of lipid A were detected in MCR-1_WT (C) and MCR-1_P198A (D) strains but not in the MCR-1_P198Y (E) strains. Unmodified lipid A species are indicated with black labels and PEA-modified species with pink labels. Representative spectra of three independent biological replicates of the genotypes are shown. (F) Purified MCR-1_P198A is resistant to cleavage by DegP. Immunoblots were developed with anti-MCR-1 MAbs. MCR-1 and its cleavage product are indicated with arrows. (G) The hydrolytic activity of MCR-1_WT, MCR-1_P198A, and MCR-1_H478A was assessed following overnight incubation of the purified enzymes solubilized in DDM with the proxy fluorescent lipid substrate acyl 12:0 NBD-glycerol-3′-phosphoethanolamine. The cleavage product acyl 12:0 NBD-glycerol was visualized using thin-layer chromatography and verified by MS analysis (see Fig. S2D). Unlike MCR-1_H478A, which was unable to catalyze PEA hydrolysis, MCR-1_P198A catalyzed the hydrolysis of PEA from the lipid substrate, similar to the wild-type enzyme MCR-1.

FIG 3

Restoration of MCR-1 expression and activity of the P198Y mutant requires ESR response components. (A to C) Growth curves of E. coli DH10β, DH10β ΔdegP, and DH10β ΔcpxRA expressing MCR-1_WT, MCR-1_P198A, or MCR-1_P198Y in LB broth supplemented with 2 mg/L colistin. The E. coli DH10β strain harboring the pP198Y plasmid is highly sensitive to colistin (A). Expression of MCR-1_P198Y in E. coli DH10β ΔdegP (B) and E. coli DH10β ΔcpxRA (C) restores colistin resistance. Data are means and SEM from three independent experiments. (D to F) Immunoblots of periplasmic fractions isolated from E. coli DH10β, E. coli DH10β ΔdegP, and E. coli DH10β ΔcpxRA expressing MCR-1_WT, MCR-1_P198A, or MCR-1_P198Y. Conversion of the cleavage site from proline to alanine (P198A) renders it resistant to DegP but has no effect on colistin resistance activity. A proline-to-tyrosine (P198Y) mutation of the cleavage site leads to loss of MCR-1 production (D). Expression of MCR-1_P198Y is restored in E. coli DH10β ΔdegP (E) and E. coli DH10β ΔcpxRA (F). (G to I) Charge-deconvoluted spectra of the MS analysis of lipid A preparations for E. coli DH10β (G; the image is the same as Fig. 2E and is included for better visualization), E. coli DH10β ΔdegP (H), and E. coli DH10β ΔcpxRA (I) expressing MCR-1_P198Y performed in the negative-ion mode and recorded in an m/z-window of 400 to 2,500. The region shown covers penta- to hepta-acylated lipid A species. Expression of MCR-1_P198Y in E. coli DH10β ΔdegP (H) or E. coli DH10β ΔcpxRA (I) restores PEA modification of lipid A. Unmodified lipid A species are indicated with black labels, and PEA-modified species are shown with pink labels. Representative spectra of three independent biological replicates of the genotypes are shown.

FIG 4

DegP introduction ameliorates MCR-1-induced stress. (A to C) Growth curves of E. coli DH10β (A), E. coli DH10β ΔdegP (B), and E. coli DH10β ΔcpxRA (C) expressing MCR-1_WT, MCR-1_P198A, MCR-1_P198Y, or MCR-1_H478A in LB broth supplemented with 100 mg/L ampicillin. MCR-1 expression imposes growth-dependent fitness costs in a ΔdegP (B) and a ΔcpxRA (C) strain. Data are means and SEM from three independent experiments. (D and E) Coexpression of DegP together with MCR-1_WT or MCR-1_H478A improves growth of E. coli DH10β lacking DegP. Growth suppression induced by MCR-1_WT and MCR-1_H478A in E. coli DH10β strains lacking DegP (B) or CpxRA (C) is reversed through reintroduction of the degP gene. Data are means and SEM from three independent experiments. (F) Induction of cpxP::lacZ activity in the E. coli PAD282 strain expressing MCR-1_WT or MCR-1_H478A is DegP dependent. To evaluate induction of the CpxRA pathway, β-galactosidase activity was measured in cpxP::lacZ reporter E. coli strains expressing WT or mutated MCR-1 variants. The strains expressing MCR-1_WT or MCR-1_H478A induced high levels of β-galactosidase activity compared to the MCR-1_P198A- or MCR-1_P198Y-producing strains. Immunoblots of periplasmic fractions show the same level of protein expression in the MCR-1_WT-, MCR-1_P198A-, or MCR-1_H478A-producing strain. Expression of DegP in the presence of MCR-1_WT, or MCR-1_H478A alleviated CpxRA pathway-dependent periplasmic stress responses. Data are means and SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 5

Growth at acidic pH induces high-level MCR-1-dependent colistin resistance. (A) Serial dilutions of E. coli DH10β expressing MCR-1_WT, MCR-1_P198A, MCR-1_P198Y, or MCR-1_H478A were spotted directly on the surface of LB agar plates supplemented with 100 mg/L ampicillin and either 0.2% or 0.8% bile salts. Plates were incubated overnight at 37°C. E. coli DH10β strains expressing active MCR-1 (MCR-1_WT or MCR-1_P198A) exhibit bile salt resistance. (B) The motility of E. coli DH10β expressing MCR-1_WT, MCR-1_P198A, MCR-1_P198Y, or MCR-1_H478A was assessed on LB agar plates supplemented with 100 mg/L ampicillin and 0.3% agar. Data are means and SEM from three independent experiments (**, P < 0.01; ***, P < 0.001). MCR-1_WT- and MCR-1_H478A-expressing isolates show reduced motility. (C) E. coli DH10β harboring pUC19::mcr-1 exhibits pH-dependent colistin resistance levels. MIC determination was performed as described in Materials and Methods. Data are representative of three independent experiments. (D) E. coli DH10β harboring pUC19::mcr-1 exhibits pH-dependent resistance to the antimicrobial peptide LL-37. Data are means and SEM from three independent experiments (**, P < 0.01; ***, P < 0.001). (E) Ratio of nonmodified (black bars) and PEA-modified (pink bars) lipid A species of E. coli DH10β pUC19 and E. coli DH10β pUC19::mcr-1 grown at pH 5 in the absence and the indicated concentrations of colistin. Calculation of the ratio of PEA-modified and unmodified lipid A is explained in Materials and Methods and can also be found in Table S2. Exemplary MS spectra are shown in Fig. S4E. (F) The hydrolytic activity of MCR-1 at different pH values was assessed by the incubation of the purified enzyme solubilized in DDM with the fluorescent lipid substrate acyl 12:0 NBD-glycerol-3′-phosphoethanolamine overnight. Incubation was carried out in buffers at different pH values as described in Materials and Methods. The cleavage product acyl 12:0 NBD-glycerol was visualized using thin-layer chromatography. MCR-1-dependent hydrolytic activity is clearly observed at pH 5 and pH 7.4 and is only marginal at pH 9. (G) Ratio of nonmodified (black bars) and PEA-modified (pink bars) lipid A species observed in the in vitro reconstitution assay of PEA modification of lipid A by MCR-1 at different pHs. MCR-1 PEA transfer activity is clearly observed at pH 5 and pH 7.4, with a significant increase at acidic pH. Calculation of the ratio of PEA-modified and unmodified lipid A was performed as described in Materials and Methods. Details can be found in Table S2. Exemplary MS spectra are shown in Fig. S5.

FIG 6

Excipient activation of DegP abrogates growth of strains harboring mcr-1 plasmids. (A to D) Excipient activation of DegP specifically eliminates strains harboring mcr-1 plasmids expressing colistin resistance. Loss of colistin resistance for the pUC-based (A) and IncX4-based (pV163M) (C) plasmids detected in the presence of colistin at various concentrations of activator peptides. Experiments were repeated with the same strains using either ampicillin (B) or kanamycin (D); resistance genes for these antibiotics are carried by pUC19 and pV163M, respectively. Here, no growth inhibition is observed. Data are means and SEM from three independent experiments.

FIG 7

Model of MCR-1-dependent activation of CpxRA processes. MCR-1 is represented with its inner membrane (IM) N-terminal transmembrane region (TM; blue), its periplasmic bridge region (PBD; green, active; red, inactive), and its C-terminal catalytic domain (CD; green, active; red, inactive). DegP (brown) and its cleavage site on MCR-1 at aa 198 are indicated. The enzyme MCR-1 hydrolyzes the substrate phosphatidylethanolamine (PE) and transfers phosphoethanolamine (PEA) to lipid A (1). During growth at neutral pH, both active and inactive (cleaved) forms of MCR-1 are present (step 2) (Fig. S4A and C). At low pH, only the active MCR-1 form predominates (Fig. S4B and D), and its activity is also enhanced (Fig. 5E to G; Fig. S4E and S5), favoring PEA modification of LPS (1). Accessibility to the conserved cleavage site activates the ESR-sensing two-component system CpxRA (3) to induce an acid-tolerant response, enabling MCR-1-producing bacteria to grow in bile salts (step 4a) (Fig. 5A), and promotes the expression of DegP, leading to the enhanced cleavage of MCR-1 (step 4b) (Fig. 1 to 4). (Core sugars and fatty acids of the LPS are only drawn symbolically and do not reflect exact structures).