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A photophysical study of the polyene antibiotic filipin. Self-aggregation and filipin--ergosterol interaction
L M Loura1, M A Castanho, A Fedorov
1Centro de Quimica-Fisica Molecular, Instituto Superior Técnico, Lisbon, Portugal. pclloura@alfa.ist.utl.pt
Abstract:
Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25 degrees C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE--filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin--filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.
Insights
Filipin antibiotic aggregates with itself and dehydroergosterol (DHE) in membranes. This suggests filipin
Area of Science:
- Biochemistry
- Membrane Biophysics
- Antimicrobial Research
Background:
- Filipin is a macrolide polyene antibiotic that interacts with ergosterol in fungal membranes.
- Understanding filipin's aggregation behavior is crucial for elucidating its mechanism of action.
Purpose of the Study:
- To investigate the aggregation of filipin and its interaction with dehydroergosterol (DHE) using fluorescence resonance energy transfer (FRET).
- To explore the implications of filipin aggregation on its membrane interaction models.
Main Methods:
- Time-resolved and steady-state fluorescence resonance energy transfer (FRET) measurements.
- Fluorescence intensity, anisotropy, and absorption spectroscopy.
- Utilized small unilamellar vesicles (SUVs) composed of dipalmitoylphosphatidylcholine (DPPC).
Main Results:
- Evidence of both DHE-filipin and filipin-filipin aggregate formation was observed.
- Filipin self-aggregation occurred even at low antibiotic concentrations (<1 mol%) and in the presence of DHE.
- Spectral changes in filipin absorption indicated a stacked arrangement in vesicles.
Conclusions:
- Filipin can form aggregates with both sterols (like DHE) and itself within membranes.
- Contradictory models of filipin action may be reconcilable, as both sterol-dependent and independent mechanisms can coexist.
- This aggregation behavior is relevant for understanding filipin's activity in low sterol environments.