The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry.

We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.

Effect of ergosterol on the interlamellar spacing of deuterated yeast phospholipid multilayers.

Sterols regulate several physico-chemical properties of biological membranes that are considered to be linked to function. Ergosterol is the main sterol molecule found in the cell membranes of yeasts and other fungi. Like the cholesterol found in mammalian cells, ergosterol has been proposed to have an ordering and condensing effect on saturated phospholipid membranes. The effects of cholesterol have been investigated extensively and result in an increase in the membrane thickness and the lipid acyl chain order. Less information is available on the effects of ergosterol on phospholipid membranes. Neutron Diffraction (ND) was used to characterize the effect of ergosterol on lipid multilayers prepared with deuterated natural phospholipids extracted from the yeast Pichia pastoris. The data show that the effect of ergosterol on membranes prepared from the natural phospholipid extract rich in unsaturated acyl chains, differs from what has been observed previously in membranes rich in saturated phospholipids. In contrast to cholesterol in synthetic phospholipid membranes, the presence of ergosterol up to 30 mol % in yeast phospholipid membranes only slightly altered the multilayer structure. In particular, only a small decrease in the multilayer d-spacing was observed as function of increasing ergosterol concentrations. This result highlights the need for further investigation to elucidate the effects of ergosterol in biological lipid mixtures.

The impact of deuteration on natural and synthetic lipids: A neutron diffraction study.

The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures.

Synthesis of Perdeuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D82 ]POPC) and Characterisation of Its Lipid Bilayer Membrane Structure by Neutron Reflectometry.

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), an unsaturated acyl chain containing lipid, is often the predominant lipid in eukaryotic cell membranes in which it is crucial for the fluidity of membranes under physiological conditions. Commercially available, partially deuterated [D31 ]1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ([D31 ]POPC) does not provide sufficient isotopic contrast for detailed structural studies of multicomponent membranes through neutron techniques. Herein, a relatively straightforward and generic chemical deuteration method is discussed for the asymmetric synthesis of perdeuterated [D31 ]1-palmitoyl-[D33 ]2-oleoyl-sn-[D5 ]glycero-[D13 ]3-phosphocholine ([D82 ]POPC) that also allows selective deuteration of any of its constituent groups. Neutron reflectivity of a [D82 ]POPC-supported bilayer was used to experimentally determine the neutron scattering length density profile of the lipid. The acyl chains of [D82 ]POPC are closely contrast-matched to heavy water, whereas the very high scattering length density of the deuterated glycerophosphocholine head groups provides good contrast to membrane-binding agents in both deuterated and non-deuterated solvent environments.