The antifungal activity of Natamycin : a novel mode of action of the polyene antibiotics

Fungal infections have recently become a growing threat to human health, especially in persons whose immune systems are compromised (for example transplant recipients or patients with HIV or cancer). Only a few effective antifungal agents are currently in use and a major problem is the increase of drug resistance. Resistance against polyene antibiotics is still a rare event, which makes these antibiotics particularly interesting as antifungal agents. Natamycin is a member of the polyene antibiotics and widely utilized in medicine for the topical treatment of fungal infections, as well as the food industry. Although this antibiotic has been used for years, the mode of action of natamycin is not known. This study has focussed on unravelling the mode of action of natamycin to gain novel insights that may lead to more effective antifungal formulations, As a starting point, the interaction of natamycin with the putative interaction partner of all polyenes, ergosterol, was examined using both in vitro (model membranes) and in vivo systems (yeast ERG mutants). These studies showed that natamycin has a specific interaction with ergosterol in membranes that requires the double bonds in the B-ring of the sterol structure. In addition, the mechanism of natamycin action in relation to membrane permeabilisation was examined using leakage assays in model membrane systems and in yeast. Surprisingly, in strong contrast to other polyenes of which the mode of action is known, natamycin did not change the permeability of any of the membranes tested. This demonstrated that natamycin acts via a different mode of action compared to other polyenes and blocks fungal growth by binding specifically to ergosterol. Because ergosterol is known to be important in the fusion and fission of membranes, the effect of natamycin on these fusion processes was examined by using an in vitro vacuolar fusion assay. It was shown that natamycin was able to inhibit vacuole fusion in vitro in an ergosterol dependent manner based on the use of different ERG deletion strains. The cellular morphology of intact yeast cells showed fragmented vacuoles after incubation with natamycin, underlining the ability of natamycin to act on ergosterol dependent fusion processes in yeast cells. The inhibition of natamycin on vacuole fusion took place at the priming phase, which indicated an inhibition of ergosterol dependent protein function. This was further examined using substrate uptake assays of plasma membrane transport proteins in yeast. Natamycin was shown to inhibit the transport of different substrates, suggesting that it is able to inhibit different plasma membrane transport proteins in fungi. Inhibition of transport by natamycin was most likely not related to a reorganization of sphingolipid-sterol domains, but may be a direct result of the disturbance of ergosterol dependent protein functions. Since other polyene antibiotics are able to interact with ergosterol to a similar extent, these findings suggest that all members of the polyene antibiotics have the basic ability to act through the inhibition of ergosterol dependent protein function