Natamycin Blocks Fungal Growth by Binding Specifically to Ergosterol without Permeabilizing the Membrane

Abstract

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.Fungal infections have recently become a growing threat to human health, especially in persons whose immune systems are compromised (for example, by human immunodeficiency virus and cancer chemotherapy). Only a few effective antifungal agents are currently in use; these include the polyenes, the fluorocytes, and the azole derivatives. One important problem is the increase of drug resistance, particularly against azole antimyotics and fluorocytosine (1). Resistance against polyene antibiotics is still a rare event, which makes these antibiotics particularly interesting as antifungal agents. The polyene antibiotics have a ring structure in which a conjugated double bond system is located opposite to a number of hydroxyl functions. Often a mycosamine group is present in combination with a carboxyl moiety, rendering the molecule amphoteric (Fig. 1). In the past convincing evidence has been presented that several members of this class of antibiotics target sterols and in particular ergosterol, the abundant and main sterol of fungal membranes (2, 3). Different types of polyene antibiotics were shown to have different modes of action despite that they share a common target. The larger polyenes like amphotericin B and nystatin form pores together with ergosterol in the plasma membrane that collapse vital ion gradients, thereby killing the cells. The smaller uncharged filipin also destroys the membrane barrier, but by a completely different mechanism. Filipin forms large complexes with sterols between the leaflets of the lipid bilayer, resulting in loss of the barrier function (2). Natamycin (also called pimaricin) is a very effective member of the polyene antibiotic family with a large standing record of applications. It is produced by Streptomyces natalensis and used against fungal infections, but it is also widely utiliz...

Polyene antibiotic that inhibits membrane transport proteins

“…This implies that these amphotericin B derivatives are also able to inhibit a broad class of essential membrane transport proteins by binding to ergosterol, thus explaining their antifungal activities. This is strengthened by the observation that both natamycin and the variant of amphotericin B of the Burke laboratory require ergosterol for interacting with the membrane and executing their antifungal activity (7,24). Amphotericin B may therefore be a good example of a polyene antibiotic that, via the binding of ergosterol, has a dual mode of action by inhibiting membrane proteins and permeabilizing the plasma membrane.…”

“…Natamycin, a member of the polyene antibiotic family, is widely used in the food industry and in pharmacotherapy for topical treatment. Unlike other polyene antibiotics, the mode of action of natamycin is not based on the ergosterol-dependent permeabilization of the plasma membrane (7). However, the immediate cessation of growth of yeasts by natamycin treatment indicates that there might be an instantaneous effect of natamycin at the level of the plasma membrane (7), which also contains the highest levels of ergosterol (8).…”

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts

“…The dramatically different electrostatic nature of selvamicin would likely preclude channel formation, with a hydrophilic yet uncharged sugar at each end of the molecule. We probed for an interaction with ergosterol using an established isothermal calorimetry assay for binding to liposome-embedded ergosterol (21,24). These experiments showed no evidence for binding, in stark contrast to control experiments using nystatin A 1 , suggesting that this interaction is much attenuated if present at all (SI Appendix, Fig.…”

New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans

“…It points to other, probably simpler mechanisms of saponin synergy with antimycotics. Natamycin belongs to the polyenes -antifungals with the rare occurrence of resistance caused mainly by a reduction in the amount of ergosterol in the plasma membrane (te Welscher et al, 2008). Therefore, we believe that the mechanism of demonstrated synergy is based on unspecific yeast cell membrane damage, which leads to its increased permeability (also for antifungal drugs), rather than blocking or modification of the efflux pumps.…”