The Antifungal Plant Defensin HsAFP1 from Heuchera sanguinea Induces Apoptosis in Candida albicans

Abstract

Plant defensins are active against plant and human pathogenic fungi (such as Candida albicans) and baker's yeast. However, they are non-toxic to human cells, providing a possible source for treatment of fungal infections. In this study, we characterized the mode of action of the antifungal plant defensin HsAFP1 from coral bells by screening the Saccharomyces cerevisiae deletion mutant library for mutants with altered HsAFP1 sensitivity and verified the obtained genetic data by biochemical assays in S. cerevisiae and C. albicans. We identified 84 genes, which when deleted conferred at least fourfold hypersensitivity or resistance to HsAFP1. A considerable part of these genes were found to be implicated in mitochondrial functionality. In line, sodium azide, which blocks the respiratory electron transport chain, antagonized HsAFP1 antifungal activity, suggesting that a functional respiratory chain is indispensable for HsAFP1 antifungal action. Since mitochondria are the main source of cellular reactive oxygen species (ROS), we investigated the ROS-inducing nature of HsAFP1. We showed that HsAFP1 treatment of C. albicans resulted in ROS accumulation. As ROS accumulation is one of the phenotypic markers of apoptosis in yeast, we could further demonstrate that HsAFP1 induced apoptosis in C. albicans. These data provide novel mechanistic insights in the mode of action of a plant defensin.

Keywords: Candida albicans; Saccharomyces cerevisiae; apoptosis; mitochondria; mode of action; plant defensin.

Figure 1

Effect of the respiration inhibitor sodium azide on HsAFP1 antifungal action. Exponentially growing S. cerevisiae cultures were suspended in PDB/YPD and incubated with 20 μg/ml HsAFP1 in the presence (black bars) or absence (white bars) of 0.005% azide for 0, 4, and 8 h of incubation at 30°C. Viability was analyzed by counting the number of CFUs/ml on YPD agar plates. Percentage survival was calculated as the ratio of the CFUs/ml after HsAFP1 treatment to the CFUs/ml of the corresponding control (water) treatment. Data represent mean ± SEM. This figure is a representative of three independent experiments.

Figure 2

HsAFP1 induces apoptosis in C. albicans. Exponentially growing C. albicans cultures were treated with 5 μg/ml HsAFP1 or water for 2 h 30 min. HsAFP1-treated cells (gray bars) and control cells (white bars) were assayed for ROS accumulation (via DHE staining), DNA fragmentation (via TUNEL staining), and phosphatidylserine externalization and membrane integrity via annexinV/propidium iodide co-staining. In each experiment, 500 cells were evaluated using fluorescence microscopy (100% represents the number of cells, i.e., 500). Values are the mean of triplicate measurements. Data represent mean ± SEM. *p < 0.05; **p < 0.01.