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Polymyxin B-phosphatidylglycerol interactions. A monolayer (pi, delta V) study
Abstract:
Through a monolayer investigation (pi, delta V), it is shown that the cationic antibiotic polymyxin B (or E) strongly interacts with films of acidic lipids, namely the didodecanoyl- and dihexadecanoylphosphatidylglycerol. The zwitterionic dihexadecanoylphosphatidylcholine was an unsuitable substrate. Interactions occurred at and above a polymyxin B concentration in the subphase of 2.5 . 10(-7) M, bringing about a considerable increase of both pi and delta V. These interactions proceeded in two steps, as revealed by a biphasic change of delta V with time. They were independent of the film molecular packing (fluid or gel states) and of the initial film pressure. Since it was possible to monitor the relative number of polymyxin B and didodecanoyl- or dihexadecanoylphosphatidylglycerol molecules in the monolayer, it is demonstrated that, at saturation, one polymyxin B molecule is bound to five phosphatidylglycerol molecules, a result which accounts for an exact neutralization of the charges. From competition experiments, it is shown that Na+ is ineffective in removing polymyxin B from the interface. Ca2+ appeared to be a stronger competitor but no complete antibiotic desorption was observed even at a Ca2+ concentration of 100 mM. As a working hypothesis, the antibiotic/lipid (1/5) system was assumed to constitute by itself one molecular species. The mixing of the polymyxin B/didodecanoylphosphatidylglycerol (1/5) system with an excess of lipid molecules in the monolayer was found to be ideal both in terms of pi and delta V. With dihexadecanoylphosphatidylglycerol, a small condensing effect could be detected only at intermediate surface pressures, in a region where the lipid phase transition occurred. The molecular area of polymyxin B interacting with didodecanoylphosphatidylglycerol can be calculated to be 1.23 +/- 0.05 nm2. It is proposed that the whole antibiotic molecule penetrates the film, the five bound lipid molecules being distributed aroung the peptide structure, at given positions imposed by the five 2,4-diaminobutyric acid residues.
Insights
The cationic antibiotic polymyxin B strongly interacts with acidic lipid films, binding to five phosphatidylglycerol molecules. This interaction, crucial for understanding antibiotic-membrane interactions, is not easily disrupted by sodium or calcium ions.
Area of Science:
- Biochemistry
- Membrane Biophysics
- Pharmacology
Background:
- Cationic antibiotics like polymyxin B are vital in combating Gram-negative bacterial infections.
- Understanding the molecular mechanisms of polymyxin B interaction with bacterial membranes is crucial for developing new therapeutic strategies.
- Lipid composition of bacterial membranes significantly influences antibiotic efficacy and resistance.
Purpose of the Study:
- To investigate the interaction of polymyxin B with model lipid monolayers composed of acidic and zwitterionic phospholipids.
- To determine the stoichiometry and biophysical characteristics of polymyxin B-lipid interactions.
- To assess the influence of cations on polymyxin B binding to lipid interfaces.
Main Methods:
- Monolayer investigation using surface pressure (pi) and surface potential (delta V) measurements.
- Interactions studied with didodecanoylphosphatidylglycerol (acidic lipid) and dihexadecanoylphosphatidylcholine (zwitterionic lipid).
- Competition experiments with Na+ and Ca2+ ions to evaluate their effect on antibiotic-lipid binding.
Main Results:
- Polymyxin B strongly interacts with acidic lipids (didodecanoyl- and dihexadecanoylphosphatidylglycerol) but not zwitterionic lipids.
- Interactions occur at polymyxin B concentrations above 2.5 x 10(-7) M, leading to increased pi and delta V.
- A 1:5 stoichiometry of polymyxin B to phosphatidylglycerol molecules was determined, indicating charge neutralization. The molecular area of bound polymyxin B was calculated as 1.23 +/- 0.05 nm2.
Conclusions:
- Polymyxin B forms a stable complex with acidic phospholipids, suggesting a primary mechanism for its membrane interaction.
- The antibiotic-lipid complex acts as a single molecular species, with ideal mixing properties observed.
- Sodium ions are ineffective in displacing polymyxin B, while calcium ions show moderate competition, highlighting the strength of the antibiotic-lipid interaction.