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Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide
Published on: December 19, 2020
Daptomycin Leakage Is Selective
Jin Zhang1, Kyle Scoten1, Suzana K Straus1
1Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
Abstract:
Daptomycin is a lipopeptide antibiotic approved for use against Gram-positive organisms, including highly resistant species. A number of studies have suggested that daptomycin kills bacteria by membrane permeabilization and depolarization. Recently a model membrane system consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) in a 1:1 ratio and the ionophore CCCP was proposed as a simple model to investigate the mode of action of daptomycin and resistance mechanisms at a molecular level. This study investigates how this model depends on the composition of the membrane and the role of CCCP. Results obtained from a fluorescence assay using pyranine show that daptomycin causes leakage in liposomes of limited stability and that CCCP promotes this leakage. A different model membrane system used here, which relies on ion selective dyes such as 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (PBFI), and 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetraammonium salt (SBFI), is a more robust alternative. Findings based on this newer model suggest that daptomycin is selective for potassium.
Insights
Daptomycin antibiotic action was investigated using model membranes. A new robust system suggests daptomycin selectively targets potassium ions, advancing understanding of its antibacterial mechanism.
Area of Science:
- Biochemistry
- Microbiology
- Membrane Biophysics
Background:
- Daptomycin is a lipopeptide antibiotic effective against Gram-positive bacteria.
- Its mechanism of action is proposed to involve membrane permeabilization and depolarization.
- Previous models used specific lipid compositions and ionophores to study daptomycin's effects.
Purpose of the Study:
- To investigate the dependence of a model membrane system on composition and the role of CCCP in studying daptomycin's mode of action.
- To evaluate a more robust model membrane system for investigating daptomycin's interaction with bacterial membranes.
- To determine the ion selectivity of daptomycin's membrane interaction.
Main Methods:
- Utilized a fluorescence assay with pyranine to assess liposome leakage.
- Employed a novel model membrane system incorporating ion-selective dyes PBFI and SBFI.
- Analyzed daptomycin's effect on membrane stability and ion permeability.
Main Results:
- Daptomycin induced leakage in liposomes of limited stability.
- The ionophore CCCP was found to enhance daptomycin-induced leakage.
- The PBFI/SBFI model demonstrated that daptomycin exhibits selectivity for potassium ions.
Conclusions:
- The previously proposed model membrane system has limitations regarding stability and CCCP dependence.
- A new model membrane system using PBFI and SBFI offers a more robust approach.
- Daptomycin's antibacterial activity is likely mediated by selective disruption of potassium ion transport across the bacterial membrane.
