Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane
- MRC Centre for Molecular Bacteriology and Infection, Imperial College London, United Kingdom
- Department of Bioengineering, Imperial College London, United Kingdom
- Department of Materials, Imperial College London, United Kingdom
- Institute of Biomedical Engineering, Imperial College London, United Kingdom
- Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, United Kingdom
- Department of Molecular Biosciences, University of Texas at Austin, United States
- National Heart and Lung Institute, Imperial College London, United Kingdom
- Department of Paediatric Respiratory Medicine, Royal Brompton Hospital, United Kingdom
Figures
Figure 1 with 6 supplements
Colistin disrupts the outer membrane but not the cytoplasmic membrane of E. coli expressing mcr-1.
(A) Quantification of LPS modified with phosphoethanolamine, expressed as the percentage of unmodified lipid A and unmodified lipid A, in whole cells and spheroplasts of E. coli MC1000 expressing mcr…
Figure 1—figure supplement 1
Colistin causes outer membrane (OM) disruption, but the process by which this leads to cytoplasmic membrane (CM) damage and bacterial lysis is not known.
Diagrammatic representation of the current hypothesised mechanism of action of colistin: (1) Colistin binds to LPS in the OM, causing displacement of cations that form bridges between LPS molecules …
Figure 1—figure supplement 2
Characterisation of the E. coli MC1000 strain harbouring a plasmid encoding the colistin resistance gene mcr-1, and an MC1000 strain containing the pDM1 plasmid only (pEmpty) as a control strain.
(A) Growth of E. coli MC1000 cells containing an empty pDM1 plasmid (pEmpty) or MC1000 cells containing the pDM1 plasmid expressing the mcr-1 gene, as determined by measuring OD600nm over 16 hr …
Figure 1—figure supplement 3
Formation of E. coli pEmpty and mcr-1 spheroplasts.
(A) Representative phase contrast micrographs of E. coli MC1000 cells harbouring an empty pDM1 plasmid (pEmpty) or a pDM1 plasmid expressing the colistin-resistance determinant mcr-1 before (Whole …
Figure 1—figure supplement 4
Conversion of E. coli whole cells to spheroplasts results in removal of the OM, and no OM contamination in the CM.
(A) Representative fluorescence microscopy images of E. coli MC1000 pEmpty cells labelled with fluorescein isothiocyanate (FITC, 0.5 mg ml−1) before (Whole Cells) and after (Spheroplasts) conversion …
Figure 1—figure supplement 5
The ratio of modified lipid A to unmodified lipid A is significantly greater in the cytoplasmic membrane (CM) than in the outer membrane (OM) of E. coli expressing mcr-1.
Representative mass spectra showing the ratio of unmodified lipid A (red) to lipid A modified with phosphoethanolamine (blue) in whole cells and spheroplasts of E. coli MC1000 expressing mcr-1, as …
Figure 1—figure supplement 6
Colistin potentiates the activity of rifampicin against colistin-resistant E. coli expressing mcr-1.
Checkerboard broth microdilution assay showing the synergistic growth-inhibitory activity of colistin and rifampicin against E. coli MC1000 cells producing MCR-1, as determined by measuring OD600nm …
Figure 2 with 2 supplements
MCR-1 protects the cytoplasmic membrane of E. coli spheroplasts from colistin but not other cationic antimicrobial peptides.
(A–C) Permeabilisation of the CM of E. coli MC1000 spheroplasts generated from bacteria expressing mcr-1 or empty plasmid control bacteria (pEmpty) during incubation with (A) colistin (4 µg ml−1), (B…
Figure 2—figure supplement 1
LPS modifications in the CM of colistin-resistant E. coli expressing mcr-1 has a small effect on membrane charge but not membrane fluidity.
(A) Fluidity of the CM of E. coli MC1000 spheroplasts producing MCR-1 or an empty plasmid control strain, as determined using the fluorescent Laurdan dye (100 µM) to generate Generalised …
Figure 2—figure supplement 2
The amount of unmodified LPS in the CM of colistin-resistant E. coli expressing mcr-1 is proportional to the degree of susceptibility to colistin-mediated CM damage.
(A) Representative mass spectra showing the presence and abundance of unmodified lipid A (red) and lipid A modified with phosphoethanolamine (blue) in MCR-1-producing spheroplasts of E. coli MC1000 …
Figure 3
Colistin damages the cytoplasmic membrane by disrupting cation bridges between LPS molecules.
(A, B) Permeabilisation of the CM of E. coli MC1000 spheroplasts generated from empty plasmid control bacteria during incubation with colistin (4 µg ml−1), in the absence or presence of either MgCl2(…
Figure 4 with 7 supplements
Murepavadin sensitises P. aeruginosa to colistin by increasing LPS abundance in the cytoplasmic membrane.
(A) Checkerboard broth microdilution assay showing the synergistic growth-inhibitory interaction between colistin and the LPS transport inhibitor murepavadin against P. aeruginosa PA14 cells, as …
Figure 4—figure supplement 1
Formation of P. aeruginosa spheroplasts.
(A, B) Representative phase contrast micrographs of P. aeruginosa PA14 cells before (A) and after (B) treatment with 0.25 mg ml−1 EDTA (to remove the OM) and 1 mg ml−1 lysozyme (to remove the cell …
Figure 4—figure supplement 2
Conversion of P. aeruginosa whole cells to spheroplasts results in removal of the OM, and no OM contamination in the CM.
(A) Representative fluorescence microscopy images of P. aeruginosa PA14 cells labelled with fluorescein isothiocyanate (FITC, 0.5 mg ml−1) before (Whole Cells) and after (Spheroplasts) conversion to …
Figure 4—figure supplement 3
Murepavadin increases the abundance of LPS in the cytoplasmic membrane (CM) of P. aeruginosa.
(A, B) Representative mass spectra (A) and quantification (B) showing the ratio of lipid A to a membrane phospholipid species (34:1,2) in the CM of P. aeruginosa PA14 spheroplasts exposed, or not, …
Figure 4—figure supplement 4
The LPS transport inhibitor murepavadin has no effect on reducing growth of P. aeruginosa at the concentration used.
Growth of P. aeruginosa PA14 cells in the presence of a sub-lethal concentration of the LptD inhibitor murepavadin at the indicated concentration, as determined by measuring OD600nm during 14 hr …
Figure 4—figure supplement 5
Polymyxin B nonapeptide (PMBN) does not display synergy with murepavadin against P. aeruginosa PA14.
Checkerboard broth microdilution assay showing the growth-inhibitory interaction between PMBN and murepavadin against P. aeruginosa PA14 cells, as determined by measuring OD600nm after 18 hr …
Figure 4—figure supplement 6
PMBN–murepavadin combination therapy does not promote killing of P. aeruginosa.
Survival of P. aeruginosa PA14 cells exposed to polymyxin B nonapeptide (1.67 µg ml−1) in the absence of presence of murepavadin (0.05 µg ml−1), as determined by c.f.u. counts (n = 4). In contrast …
Figure 4—figure supplement 7
Murepavadin enhances the ability of colistin to damage the CM and trigger bacterial lysis.
(A) Permeabilisation of the CM of P. aeruginosa PA14 spheroplasts by colistin (2 µg ml−1) following exposure, or not, of bacteria to murepavadin (0.05 µg ml−1) for 2 hr prior to conversion to …
Figure 5 with 2 supplements
Colistin-murepavadin combination therapy promotes killing of P. aeruginosa in vitro and in vivo.
(A) Survival of P. aeruginosa PA14 cells exposed to colistin (2 µg ml−1) in the absence of presence of murepavadin (0.05 µg ml−1), as determined by c.f.u. counts (n = 4; *p<0.05 for colistin and …
Figure 5—figure supplement 1
Murepavadin potentiates the activity of sub-lethal colistin concentrations, leading to enhanced CM damage, cell lysis and bacterial killing.
(A) OM disruption of P. aeruginosa PA14 cells during 10 min exposure to colistin (1 μg ml−1) in the absence or presence of murepavadin (0.05 μg ml−1), as assessed by uptake of the fluorescent dye …
Figure 5—figure supplement 2
Antibiogram summarising the antimicrobial susceptibilities of a panel of MDR P. aeruginosa human clinical strains isolated from sputum samples of cystic fibrosis patients.
S: susceptible (green), I: intermediate (orange), R: resistant (red). Bacterial strains were isolated and antimicrobial susceptibility testing was performed at the Royal Brompton Hospital, Royal …
Figure 6
Colistin kills bacteria by targeting LPS in both the outer and cytoplasmic membrane (CM), leading to disruption of the cell envelope and bacterial lysis.
Diagrammatic representation of the novel proposed mechanism of action of colistin: Colistin binds to LPS in the OM (1), displacing cations that form bridges between LPS molecules, which leads to …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | MC1000 | Dortet et al., 2018 PMID:30442963 | pEmpty | Background strain (araD139, ∆(ara, leu)7697, ∆lacX74, galU, galK, strA) harbouring the IPTG-inducible pDM1 plasmid (GenBank MN128719) |
| Strain, strain background (Escherichia coli) | MC1000 | Dortet et al., 2018 PMID:30442963 | mcr-1 | MC1000 strain harbouring the pDM1 plasmid encoding the mcr-1 gene amplified from a clinical E. coli isolate |
| Strain, strain background (Pseudomonas aeruginosa) | PA14 | Lee et al., 2006 PMID:17038190 | PA14 | Wild-type reference strain; highly virulent human isolate representing most common clonal group worldwide |
| Strain, strain background (Pseudomonas aeruginosa) | AK3 | This study | AK3 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient – mucoid strain |
| Strain, strain background (Pseudomonas aeruginosa) | AK10 | This study | AK10 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK20 | This study | AK20 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK6 | This study | AK6 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient – mucoid strain |
| Strain, strain background (Pseudomonas aeruginosa) | AK12 | This study | AK12 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient – mucoid strain |
| Strain, strain background (Pseudomonas aeruginosa) | AK8 | This study | AK8 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient – mucoid strain |
| Strain, strain background (Pseudomonas aeruginosa) | AK17 | This study | AK17 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK9 | This study | AK9 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK14 | This study | AK14 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK5 | This study | AK5 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient – mucoid strain |
| Strain, strain background (Pseudomonas aeruginosa) | AK11 | This study | AK11 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient – mucoid strain |
| Strain, strain background (Pseudomonas aeruginosa) | AK13 | This study | AK13 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK18 | This study | AK18 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK22 | This study | AK22 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient |
| Strain, strain background (Pseudomonas aeruginosa) | AK19 | This study | AK19 | Multi-drug resistant human clinical isolate from sputum of cystic fibrosis patient – murepavadin-resistant |
| Chemical compound, drug | Colistin | Sigma-Aldrich | C4461-1G | Targets LPS |
| Chemical compound, drug | Murepavadin (POL7080) | DC Chemicals | DC11273 | Targets LptD |
| Chemical compound, drug | Daptomycin | Bio-Techne Ltd | 3917/10 | Targets phosphatidylglycerol in the bacterial membrane |
| Chemical compound, drug | Nisin | Sigma-Aldrich | N5764-5G | Targets bacterial membranes |
| Chemical compound, drug | Rifampicin | Molekula Ltd | 32609202 | Targets RNA Polymerase |
| Chemical compound, drug | Polymyxin B nonapeptide (PMBN) | Sigma-Aldrich | P2076-5MG | Permeabilises the OM |
| Chemical compound, drug | Tetracycline | Sigma-Aldrich | 87128–25G | Protein synthesis inhibitor |
| Commercial assay or kit | LAL Chromogenic Endotoxin Quantitation Kit | Thermo Scientific Pierce | 88282 | Quantitative assay for LPS |
| Chemical compound, drug | Isopropyl β-d-1-thiogalactopyranoside (IPTG) | Melford Laboratories | MB1008 | Induces gene expression |
| Chemical compound, drug | Lysozyme from chicken egg white | Sigma-Aldrich | L6876-1G | Degrades peptidoglycan |
| Chemical compound, drug | Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | E6511-100G | Removes OM from bacteria |
| Chemical compound, drug | Trypsin | Sigma-Aldrich | T7309-1G | Degrades proteins |
| Chemical compound, drug | Propidium iodide (PI) | Sigma-Aldrich | P4864-10ML | Fluoresces when bound to DNA |
| Chemical compound, drug | Fluorescein 5 (6)-isothiocyanate | Sigma-Aldrich | 46950–50MG-F | Used to label proteins with a fluorescent tag |
| Chemical compound, drug | N-phenyl-1-napthylamine (NPN) | Acros Organics | 147160250 | Fluoresces when bound to phospholipids |
| Chemical compound, drug | FITC-labelled Poly-L-lysine (PLL) | Sigma-Aldrich | P3543-10MG | Used to measure membrane surface charge |
| Chemical compound, drug | 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan) | Sigma-Aldrich | 40227–100 MG | Used to measure membrane fluidity |
Additional files
-
Supplementary file 1
Contains Supplementary Table 1 detailing the minimum inhibitory concentrations (MICs) of colistin and murepavadin against the P. aeruginosa strains used in this study.
- https://cdn.elifesciences.org/articles/65836/elife-65836-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/65836/elife-65836-transrepform-v2.docx
