Natamycin sequesters ergosterol and interferes with substrate transport by the lysine transporter Lyp1 from yeast

Szomek, Maria; Reinholdt, Peter; Walther, Hanna-Loisa; Scheidt, Holger A.; Müller, Peter; Obermaier, Sebastian; Poolman, Bert; Kongsted, Jacob; Wüstner, Daniel

doi:10.1016/j.bbamem.2022.184012

Cited by 13 publications

(38 citation statements)

References 94 publications

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“…This could indicate preferred binding to some membrane areas or formation of natamycin aggregates in the PM. Supporting an aggregation model, we found previous evidence for the formation of natamycin oligomers in aqueous solution and model lipid membranes based on alterations in the fluorescence spectrum and on molecular dynamics (MD) simulations 17 . Here, we observe that after 2h of incubation of yeast with natamycin, lateral intensity peaks were pronounced resulting in heterogeneous PM staining (Fig.…”

Section: Resultssupporting

confidence: 59%

“…Using NMR spectroscopy of deuterated sterols and MD simulations, we demonstrated recently that natamycin immobilizes both sterols in model membranes to a similar extent 18 . At the same time, we observed that natamycin interacts preferentially with ergosterol-containing model membranes interfering with lipid phase properties and transport activity of reconstituted amino acid transporters 17,18 . Based on these findings, we conclude that the preferred interaction of natamycin with ergosterol-containing yeast must be due to specific properties of the PM mediated by the yeast sterol, which cannot be replaced completely by mammalian cholesterol.…”

Section: Discussionmentioning

confidence: 78%

“…Using a synchrotron as source of X-rays in the appropriate energy range of about 510 eV and suitable zone plates to focus the incident X-rays to the sample, SXT can obtain 30-50 nm isotropic resolution with typical acquisition times of about 30 min per tomogram 42,43 . Due to their high abundance of carbon, natamycin aggregates forming in solution above the critical aggregation concentration of the polyene 17 can be easily visualized by SXT (Fig. S3).…”

Section: Resultsmentioning

confidence: 99%

“…Mixing polyenes with sterols in water indeed causes formation of sterol-polyene complexes 17,23,29,30 , but this could also interfere with initial polyene binding to the yeast membrane as well as with sterol extraction into extracellular polyene sponges. AmpB and nystatin rapidly bind to model membranes, and we have recently shown that this is also the case for natamycin 17 . Initial binding is followed by slower membrane rearrangements including self-aggregation of the polyenes in the bilayer.…”

Section: Introductionmentioning

confidence: 99%

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Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Szomek,

Akkerman,

Lauritsen

et al. 2023

Preprint

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Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

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Section: Resultssupporting

confidence: 59%

Section: Discussionmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Szomek,

Akkerman,

Lauritsen

et al. 2023

Preprint

Self Cite

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“…Natamycin (NA) is a naturally occurring polyene macrolide antimicrobial agent generated by Streptomyces natalensis (Aparicio et al, 2016; Koontz et al, 2003). The NA is elucidated to bind irreversibly to ergosterol, form specific complexes in the bilayer and interfere with the transport of Lyp1‐transporter across the plasma membrane, thereby further suppressing the growth of yeast and fungus (Duran et al, 2016; Szomek et al, 2022). The oxidation of hydrogen peroxide destroys the membrane lipids, protein structure, as well as DNA of microorganisms by engendering cytotoxic oxidants species (OH• and O 2• − ), rapidly inducing cytoplasmic disintegration of pathogenic bacteria (de Siqueira Oliveira et al, 2018; Linley et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Combination of ethrel and antiseptic medium flow of micro‐circulating improves shelf‐life quality of kiwifruit after long‐term controlled atmosphere storage

Ran

Zheng

et al. 2022

Food Processing Preservation

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The uneven maturity and severe internal rot of kiwifruit were rapidly accelerated when transferred to room temperature after long‐term controlled atmosphere storage. In this study, the effects of ethrel combined with different flow microcirculation of antiseptic mediums on the shelf‐life quality of “Qinmei” kiwifruit stored at 20°C were evaluated. Results indicated that the combined aerosolization application of 500 mg/L natamycin, 7.5% w/w H2O2 and 0.5 mg/L atomized ozone could control the decay incidence and respiratory rate of ethrel‐treated kiwifruit. Further studies indicated that ethrel and O3 treatment greatly increased the activities of chitinase, β‐1,3‐glucanase, catalase, and superoxide dismutase and produced higher ascorbic acid levels (151.16 mg·100 g−1/FW) to enhance disease‐resistant of kiwifruit. This treatment could inhibit the activity of polyphenol oxidase and delay the process of membrane lipid peroxidation, contributing to preserving the integrity of cell structures well. Moreover, ethrel + O3 treatment could effectively reduce ethylene production (3.12 μl/kg/h) to delay the ripening and senescence of fruit stored at 20°C. More importantly, this treatment exhibited the lowest contents of decay incidence (9.75%), total bacterial count (4.07 log cfu/g), and yeasts and molds (4.18 log cfu/g), and thus extended the shelf life for up to 16 days. Therefore, a combination of ethrel and ozone is a potentially effective method for the maintenance of the shelf‐life properties of kiwifruit after long‐term cold storage. Novelty impact statement In this study, the treatment of ethrel‐treated with aerosolized ozone of flow microcirculation of 0.5 mg/L O3 could effectively suppress ripening and senescence, enhance antioxidant capacity and disease resistance, reduce the degree of membrane lipid peroxidation, and considerably preserve the integrity of cell structures, which contributed to preventing the penetration of pathogens, retarding the decay incidence, and delaying the shelf‐life quality deterioration of kiwifruit transferred to room temperature after long‐term controlled atmosphere storage.

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