Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer.

Wojciech Grudzinski,Rafal Luchowski,Wieslaw I Gruszecki,Renata Welc,Joanna Sagan

doi:10.1038/srep32780

Abstract

Amphotericin B is a popular antifungal antibiotic, a gold standard in treatment of systemic mycotic infections, due to its high effectiveness. On the other hand, applicability of the drug is limited by its considerable toxicity to patients. Biomembranes are a primary target of physiological activity of amphotericin B and both the pharmacologically desired and toxic side effects of the drug relay on its molecular organization in the lipid phase. In the present work, molecular organization, localization and orientation of amphotericin B, in a single lipid bilayer system, was analysed simultaneously, thanks to application of a confocal fluorescence lifetime imaging microscopy of giant unilamellar vesicles. The results show that the presence of sterols, in the lipid phase, promotes formation of supramolecular structures of amphotericin B and their penetration into the membrane hydrophobic core. The fact that such an effect is substantially less pronounced in the case of cholesterol than ergosterol, the sterol of fungal membranes, provides molecular insight into the selectivity of the drug.

Highlights

Owing to a dramatic increase in systemic fungal infections, in particular associated with the HIV pandemic and organ transplantation, effective treatment of fungal infections is an important and topical issue[1,2]
In the present work we readdress the problem of molecular organization, localization and orientation of amphotericin B (AmB) molecules with respect to sterol-free and sterol-containing lipid membranes, with application of very well defined experimental system consisting of a single lipid bilayer and with application of ultrasensitive confocal fluorescence lifetime imaging microscopy (FLIM) technique
In the present work we developed an experimental approach which enables combined and simultaneous analysis of the molecular organization and orientation of AmB molecules with respect to a single lipid bilayer membrane

Summary

Introduction

Owing to a dramatic increase in systemic fungal infections, in particular associated with the HIV pandemic and organ transplantation, effective treatment of fungal infections is an important and topical issue[1,2]. Several molecular mechanisms have been proposed to be directly associated with the physiological activity of AmB in biomembranes Among these mechanisms the most popular is formation of pores, independently in two lipid monolayers of a bilayer, assembly of which may yield transmembrane channels[5]. In the present work we readdress the problem of molecular organization, localization and orientation of AmB molecules with respect to sterol-free and sterol-containing lipid membranes, with application of very well defined experimental system consisting of a single lipid bilayer and with application of ultrasensitive confocal fluorescence lifetime imaging microscopy (FLIM) technique. The experimental model applied has been tested on single giant unilamellar vesicles (GUV) containing popular fluorescence probe Nile blue (NB, Fig. S2)

Methods

Results

Conclusion