Akkerman Vibeke, Scheidt Holger A, Reinholdt Peter, Bashawat Mohammad, Szomek Maria, Lehmann Max, Wessig Pablo, Covey Douglas F, Kongsted Jacob, Müller Peter, Wüstner Daniel
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230, Odense M, Denmark.
Institute for Medical Physics and Biophysics, Leipzig University, Härtelstr. 16-18, D-04107, Leipzig, Germany.
BBA Adv. 2023 Aug 25;4:100102. doi: 10.1016/j.bbadva.2023.100102. eCollection 2023.
Natamycin is an antifungal polyene macrolide that is used as a food preservative but also to treat fungal keratitis and other yeast infections. In contrast to other polyene antimycotics, natamycin does not form ion pores in the plasma membrane, but its mode of action is poorly understood. Using nuclear magnetic resonance (NMR) spectroscopy of deuterated sterols, we find that natamycin slows the mobility of ergosterol and cholesterol in liquid-ordered (Lo) membranes to a similar extent. This is supported by molecular dynamics (MD) simulations, which additionally reveal a strong impact of natamycin dimers on sterol dynamics and water permeability. Interference with sterol-dependent lipid packing is also reflected in a natamycin-mediated increase in membrane accessibility for dithionite, particularly in bilayers containing ergosterol. NMR experiments with deuterated sphingomyelin (SM) in sterol-containing membranes reveal that natamycin reduces phase separation and increases lipid exchange in bilayers with ergosterol. In ternary lipid mixtures containing monounsaturated phosphatidylcholine, saturated SM, and either ergosterol or cholesterol, natamycin interferes with phase separation into Lo and liquid-disordered (Ld) domains, as shown by NMR spectroscopy. Employing the intrinsic fluorescence of natamycin in ultraviolet-sensitive microscopy, we can visualize the binding of natamycin to giant unilamellar vesicles (GUVs) and find that it has the highest affinity for the Lo phase in GUVs containing ergosterol. Our results suggest that natamycin specifically interacts with the sterol-induced ordered phase, in which it disrupts lipid packing and increases solvent accessibility. This property is particularly pronounced in ergosterol containing membranes, which could underlie the selective antifungal activity of natamycin.
那他霉素是一种抗真菌的多烯大环内酯类药物，用作食品防腐剂，也用于治疗真菌性角膜炎和其他酵母菌感染。与其他多烯抗真菌药不同，那他霉素不会在质膜中形成离子孔，但其作用方式尚不清楚。通过对氘代固醇进行核磁共振（NMR）光谱分析，我们发现那他霉素在液态有序（Lo）膜中使麦角固醇和胆固醇的流动性减慢程度相似。分子动力学（MD）模拟也支持了这一点，该模拟还揭示了那他霉素二聚体对固醇动力学和水渗透性的强烈影响。对固醇依赖性脂质堆积的干扰也反映在那他霉素介导的连二亚硫酸盐膜可及性增加上，特别是在含有麦角固醇的双层膜中。在含固醇的膜中对氘代鞘磷脂（SM）进行的NMR实验表明，那他霉素可减少相分离并增加含麦角固醇双层膜中的脂质交换。如NMR光谱所示，在含有单不饱和磷脂酰胆碱、饱和SM以及麦角固醇或胆固醇的三元脂质混合物中，那他霉素会干扰相分离成Lo和液态无序（Ld）结构域。利用那他霉素在紫外敏感显微镜下的固有荧光，我们可以观察到那他霉素与巨型单层囊泡（GUV）的结合，发现它对含有麦角固醇的GUV中的Lo相具有最高亲和力。我们的结果表明，那他霉素与固醇诱导的有序相特异性相互作用，在该相中它破坏脂质堆积并增加溶剂可及性。这种特性在含麦角固醇的膜中尤为明显，这可能是那他霉素选择性抗真菌活性的基础。
