Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven Heverlee, Belgium.
Front Microbiol. 2011 Mar 16;2:47. doi: 10.3389/fmicb.2011.00047. eCollection 2011.
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.
植物防御素对植物和人类病原真菌（如白色念珠菌）和面包酵母具有活性。然而，它们对人类细胞无毒，为治疗真菌感染提供了可能的来源。在这项研究中，我们通过筛选酿酒酵母缺失突变体文库，确定了珊瑚铃植物防御素 HsAFP1 的作用模式，该文库中的突变体对 HsAFP1 的敏感性发生了改变，并通过酿酒酵母和白色念珠菌中的生化分析验证了获得的遗传数据。我们鉴定了 84 个基因，当这些基因缺失时，至少会使酵母对 HsAFP1 表现出四倍以上的超敏或抗性。这些基因中有相当一部分被发现与线粒体功能有关。与此一致的是，阻断呼吸电子传递链的叠氮化钠拮抗了 HsAFP1 的抗真菌活性，这表明功能性呼吸链对于 HsAFP1 的抗真菌作用是必不可少的。由于线粒体是细胞活性氧（ROS）的主要来源，我们研究了 HsAFP1 诱导 ROS 的性质。我们表明，HsAFP1 处理白色念珠菌会导致 ROS 积累。由于 ROS 积累是酵母细胞凋亡的表型标记之一，我们可以进一步证明 HsAFP1 诱导了白色念珠菌的凋亡。这些数据为植物防御素的作用模式提供了新的机制见解。
