Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, United Kingdom.
PLoS One. 2012;7(9):e43989. doi: 10.1371/journal.pone.0043989. Epub 2012 Sep 13.
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 Ca(2+) elevations. We investigated the eugenol Ca(2+) signature in further detail and show that exponentially growing cells exhibit Ca(2+) elevation resulting exclusively from the influx of Ca(2+) across the plasma membrane whereas in stationary growth phase cells Ca(2+) influx from intracellular and extracellular sources contribute to the eugenol-induced Ca(2+) elevation. Ca(2+) channel deletion yeast mutants were used to identify the pathways mediating Ca(2+) influx; intracellular Ca(2+) release was mediated by the vacuolar Ca(2+) channel, Yvc1p, whereas the Ca(2+) 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 Ca(2+) channel, Cch1p, was hypersensitive to eugenol and that this correlated with reduced Ca(2+) elevations. Taken together, these results indicate that a cch1p-mediated Ca(2+) 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.
丁香酚具有抗真菌活性，被认为具有治疗潜力。然而，人们对其抗真菌活性的细胞基础知之甚少，更好地了解丁香酚的耐受性应该会导致更好地利用丁香酚进行抗真菌治疗。使用表达脱辅基水母发光蛋白的模式酵母酿酒酵母来表明丁香酚诱导细胞溶质 Ca(2+) 升高。我们进一步详细研究了丁香酚 Ca(2+) 特征，表明指数生长期细胞表现出 Ca(2+) 升高，这仅源自质膜的 Ca(2+) 内流，而在静止生长阶段，Ca(2+) 内流来自细胞内和细胞外来源有助于丁香酚诱导的 Ca(2+) 升高。使用 Ca(2+) 通道缺失酵母突变体来鉴定介导 Ca(2+) 内流的途径；细胞内 Ca(2+) 释放由液泡 Ca(2+) 通道 Yvc1p 介导，而质膜 Ca(2+) 内流可分为 Cch1p 依赖和 Cch1p 非依赖途径。我们表明缺乏质膜 Ca(2+) 通道 Cch1p 的酵母生长对丁香酚敏感，这与 Ca(2+) 升高减少有关。总之，这些结果表明，Cch1p 介导的 Ca(2+) 内流是一种细胞内信号的一部分，可防止丁香酚毒性。这项研究为真菌耐受丁香酚毒性所采用的机制提供了新的见解，这应该会导致更好地利用丁香酚进行抗真菌治疗。
