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This study aimed to characterize the antifungal effects of chiloscyphenol A (CA), a natural small molecule isolated from Chinese liverworts, and investigate its mode of action. CA was effective against five tested  Candida  species with a minimal inhibitory concentration (MIC) of 8–32 μg/ml and exhibited fungicidal activity against  Candida albicans  in both the planktonic state and mature biofilms. The  in vivo  study using  Caenorhabditis elegans  showed that CA prolonged the survival of  C. albicans  infected worms. Further investigations revealed that CA resulted in mitochondrial dysfunction as indicated by mtΔψ hyperpolarization, increased ATP production and intracellular ROS accumulation, and aggregated distribution of Tom70. In addition, CA caused perturbation of the cell membrane and increased membrane permeability, as demonstrated by specific staining and confocal microscopic and transmission electron microscopy (TEM) observations and by calcein-leakage measurements. This conclusion was further confirmed by the decreased cell size of CA-treated cells via three-dimensional contour-plot analysis using flow cytometry. Taken together, these results suggest that CA exerts fungicidal activity by eliciting both mitochondrial dysfunction and plasma membrane destruction in  C. albicans . The elucidated mechanism supports the potential application of CA against clinical fungal infections.

Chiloscyphenol A derived from Chinese liverworts exerts fungicidal action by eliciting both mitochondrial dysfunction and plasma membrane destruction