Abstract
Eugenol has antifungal activity and is recognised as having therapeutic potential. However, little is known of the cellular basis of its antifungal activity and a better understanding of eugenol tolerance should lead to better exploitation of eugenol in antifungal therapies. The model yeast, Saccharomyces cerevisiae, expressing apoaequorin was used to show that eugenol induces cytosolic Ca2+ elevations. We investigated the eugenol Ca2+ signature in further detail and show that exponentially growing cells exhibit Ca2+ elevation resulting exclusively from the influx of Ca2+ across the plasma membrane whereas in stationary growth phase cells Ca2+ influx from intracellular and extracellular sources contribute to the eugenol-induced Ca2+ elevation. Ca2+ channel deletion yeast mutants were used to identify the pathways mediating Ca2+ influx; intracellular Ca2+ release was mediated by the vacuolar Ca2+ channel, Yvc1p, whereas the Ca2+ influx across the plasma membrane could be resolved into Cch1p-dependent and Cch1p-independent pathways. We show that the growth of yeast devoid the plasma membrane Ca2+ channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca2+ elevations. Taken together, these results indicate that a cch1p-mediated Ca2+ influx is part of an intracellular signal which protects against eugenol toxicity. This study provides fresh insight into the mechanisms employed by fungi to tolerate eugenol toxicity which should lead to better exploitation of eugenol in antifungal therapies.
Highlights
Fungi are emerging as major causes of human infections, amongst a growing population of immunocompromised hosts, which is having significant economic and social impacts [1]
Possible modes of action to explain the antifungal capacity of these compounds have been suggested, including general disruption of membrane integrity and consequential disruption of cell signalling and leakage of cell contents [4]; the mechanism of killing is not clear and we know nothing about the mechanisms employed by fungi to resist the antifungal properties of plant essential oils
Despite these new insights, it remains unclear if the Ca2+cyt elevation induced by these plant phenolic compounds represents an antifungal activity or if it forms part of signalling response to protect against the fungicidal activity
Summary
Fungi are emerging as major causes of human infections, amongst a growing population of immunocompromised hosts, which is having significant economic and social impacts [1]. We explored the possibility that Cch1p and Mid1p mediated eugenol induced Ca2+ influx across the plasma membrane using Ca2+ channel mutant yeast strains transformed with pEVP11/ AEQ [8] resulting in cytosolic expression of the Ca2+-sensitive bioluminescent protein aequorin.
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