We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.

• MeSH
• Color MeSH
• Cholesterol chemistry   MeSH
• Dimerization MeSH
• Microscopy, Fluorescence methods   MeSH
• Spectrometry, Fluorescence MeSH
• Membrane Fusion * MeSH
• Microscopy, Confocal MeSH
• Lipids chemistry   MeSH
• Lipopeptides chemistry   MeSH
• Peptides chemistry   MeSH
• Polysorbates chemistry   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Unilamellar Liposomes chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: alphahemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size - 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities alpha for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV(1000)) were found to be more suitable for distinguishing between high alpha values whereas smaller ones (LUV(100)) were more appropriate for discriminating an all-or-none leakage (alpha=0) from the graded leakage with low values of alpha. While disrupting LUV(1000), CyaA caused a highly cation-selective leakage (alpha~15) whereas its mutated form with decreased channel K(+)/Cl(-) selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K+E516K) exhibited much lower selectivity (alpha approximately 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.

• MeSH
• Adenylate Cyclase Toxin metabolism   MeSH
• Bordetella pertussis physiology   MeSH
• Cell Membrane physiology   MeSH
• Electrophoresis, Agar Gel MeSH
• Escherichia coli physiology   MeSH
• Spectrometry, Fluorescence MeSH
• Hemolysin Proteins metabolism   MeSH
• Kinetics MeSH
• Liposomes metabolism   MeSH
• Cell Membrane Permeability physiology   MeSH
• Escherichia coli Proteins metabolism   MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH