Although the biological consequences of the action of the polyene antifungal compound natamycin are not known, the mode of action is thought to arise via a specific interaction with ergosterol but does not involve membrane permeabilization. In this study, we have demonstrated that natamycin is able to interfere in the process of vacuole fusion in a sterol-dependent manner. This inhibition also did not involve membrane permeabilization and seemed to take place early in the fusion mechanism, even before any membrane contact had occurred.
Ergosterol is known to be important during fusion and fission processes, including vacuole fusion (12
). To determine if natamycin was able to act on these processes via its specific interaction with ergosterol, the effects of this antibiotic on the fusion of isolated yeast vacuoles were studied using a content mixing assay (15
). Indeed, natamycin was shown to inhibit the fusion process of isolated vacuoles. In addition, this inhibition was not related to a permeabilizing effect, similar to natamycin's inability to permeabilize model membranes or yeast cells (32
). The sterol structure dependency of the vacuolar fusion inhibition by natamycin was almost identical to the sterol structure dependency for its activity toward yeast cells and its binding to sterols in model membranes (32
). All were dependent on the presence of sterols containing double bonds in the B-ring, most importantly at the 7,8 position (32
). Therefore, we conclude that natamycin inhibits vacuolar fusion through the specific interaction with ergosterol.
Treatment of yeast cells with natamycin led to a fragmented vacuolar morphology that is characteristic of a defect in vacuole fusion (1
). A similar vacuolar morphology has been observed in conidia of Penicillium discolor
upon natamycin treatment (M. R. van Leeuwen and J. Dijksterhuis, unpublished observations). We therefore conclude that natamycin is able to inhibit vacuole fusion both in purified vacuoles and in intact yeast cells. Besides this inhibition of vacuolar fusion, natamycin may act on more ergosterol-dependent membrane fusion and fission processes through its interaction with ergosterol (23
). Indeed, natamycin has been shown to inhibit the early stages of endocytosis in the fungus P. discolor
), an ergosterol-dependent fission process (17
). This suggests that the basis of the toxicity of natamycin could be the inhibition of fusion and fission processes. To act on vacuole fusion in an intact yeast cell most likely requires natamycin to enter this cell. This could be either via permeation across the plasma membrane or in an early stage via endocytosis. Currently we have no information on whether natamycin enters the cell and if so via which mechanism this occurs.
The polyene antibiotics nystatin and filipin were shown to be more efficient in inhibiting the fusion of isolated vacuoles. These differences are probably directly related to the relative affinity of the polyenes for ergosterol and their differences in membrane-permeabilizing activity. Nystatin and natamycin had binding affinities similar to those of ergosterol (32
), yet nystatin is more efficient in its inhibition of vacuole fusion. This is best explained by the ability of nystatin to permeabilize the vacuole membrane, thereby increasing its efficacy of vacuole fusion inhibition. Filipin displayed the highest affinity for ergosterol, and it severely damages the membrane barrier (10
). Altogether, this likely explains why filipin was the most efficient inhibitor of vacuole fusion in our assays.
What would be the mechanism behind the inhibition of fusion caused by natamycin? We have observed that through the specific interaction with ergosterol, natamycin was able to act on the early priming stage of fusion. During this phase, no actual contact between the vacuolar membranes has taken place (25
), making it unlikely that natamycin will act on lipid reorganization. The priming phase consists solely of the rearrangements of different protein complexes (for reviews, see references 25
). Thus, the most straightforward conclusion is that natamycin is able to disturb these rearrangements as a result of its binding to ergosterol, and this suggests a more general mode of action, namely, to disturb ergosterol-dependent protein functions.
This immediately poses the question of whether the other members of the family of polyene antibiotics, which all bind to ergosterol (3
), are also able to act on the priming stage through their interaction with ergosterol. Indeed, we have shown that nystatin is able to act on the priming stage, as was observed previously as well, and the same is true for amphotericin B (21
). The effect of filipin is less clear, because we found it to act in between the docking and fusion stages, while in a different study filipin was shown to act on the priming stage (21
). The differences in results are likely best explained by different assay conditions. These findings points to a dual mode of action for some members of the polyene antibiotic family, where all members have the basic ability to act through the inhibition of ergosterol-dependent protein functions, while the additional ability is to permeabilize the membrane. This relates to a freeze fracture electron microscopy study, where natamycin, nystatin, and filipin all produced distinct morphological effects on the fungal membrane, indicating the different end results according to the mechanisms involved in polyene-sterol interactions (22
). Because natamycin has only the basic ability to bind ergosterol, it is the ideal candidate for studying the basic mode of action of the polyenes. In addition, this makes natamycin an interesting tool for cell biology when analyzing ergosterol-dependent protein functions.
Interestingly, there is another naturally produced family of antibiotics where several members are known to have a dual mode of action. This is the antibacterial lantibiotic family, a group of small antimicrobial peptides, a large part of which are known to bind the bacterial cell wall component lipid II and through this interaction block cell wall synthesis (5
). In the group of lantibiotics that are able to bind lipid II, several members are long enough to span the lipid bilayer and have an additional ability to form pores (5
). This striking parallel shows how nature has repeatedly used dual modes of action for membrane-active antibiotics, and this might be applicable to other families of membrane active antibiotics as well.