Lipid polyunsaturation determines the extent of membrane structural changes induced by Amphotericin B in Pichia pastoris yeast

Abstract

The activity of the potent but highly toxic antifungal drug Amphotericin B (AmB), used intravenously to treat systemic fungal and parasitic infections, is widely accepted to result from its specific interaction with the fungal sterol ergosterol. While the effect of sterols on AmB activity has been intensely investigated, the role of membrane phospholipid composition has largely been ignored, and structural studies of native membranes have been hampered by their complex and disordered nature. We show for the first time that the structure of fungal membranes derived from Pichia pastoris yeast depends on the degree of lipid polyunsaturation, which has an impact on the structural consequences of AmB activity. AmB inserts in yeast membranes even in the absence of ergosterol, and forms an extra-membraneous layer whose thickness is resolved to be 4-5 nm. In ergosterol-containing membranes, AmB insertion is accompanied by ergosterol extraction into this layer. The AmB-sponge mediated depletion of ergosterol from P. pastoris membranes gives rise to a significant membrane thinning effect that depends on the degree of lipid polyunsaturation. The resulting hydrophobic mismatch is likely to interfere with a much broader range of membrane protein functions than those directly involving ergosterol, and suggests that polyunsaturated lipids could boost the efficiency of AmB. Furthermore, a low degree of lipid polyunsaturation leads to least AmB insertion and may protect host cells against the toxic effects of AmB. These results provide a new framework based on lipid composition and membrane structure through which we can understand its antifungal action and develop better treatments.

The Production of Matchout-Deuterated Cholesterol and the Study of Bilayer-Cholesterol Interactions

“…The availability of deuterated lipids is crucial for neutron scattering studies that exploit the very large difference in the scattering length densities ( ρ ) of protium and deuterium. This approach provides a way to study the positioning of a lipid molecule with respect to different environments and has successfully been used for the study of several systems including mixtures of deuterated phospholipids 15–17 and unlabelled sterols 18 . The deployment of D 2 O matched-deuteration, referred to herein as simply matchout-deuteration, (in contrast to per deuteration) in neutron structural studies has been discussed recently by Dunne et al .…”

Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids

“…However, complex lipid mixtures of both hydrogenated and deuterated yeast extracts have recently been successfully deposited and characterized, showing a clear advancement in this field. [ 16 ]…”

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

“…The advantages lie in obtaining different contrasts, which can be controlled through different deuteration levels of the different parts of the lipid; thus highlighting specific parts. The use of such chemically synthesised lipids with a well‐defined molecular structure also provides a means of investigating the effects of individual molecular species on membrane interactions, which is challenging in complex biological lipid mixtures derived from cell cultures . This can then be exploited in studies of membrane proteins and potential drugs, as well as their interactions with the membrane.…”

“…The use of such chemically synthesised lipids with aw ell-defined molecular structure also provides am eans of investigating the effects of individual molecular species on membrane interactions, which is challenging in complex biological lipid mixtures derivedf rom cell cultures. [46] This can then be exploited in studies of membrane proteins and potential drugs, as well as their interactions with the membrane. In this context, the use of perdeuteratedl ipids such as 8 to understand in more detail,for example, the interactions of antimicrobialcompounds with bacterial membrane mimics, [47] could lead to novel insights into the increasing problem of antibiotic resistance in bacteria.…”