The Antifungal Plant Defensin HsAFP1 from Heuchera Sanguinea Induces Apoptosis in Candida Albicans

An M Aerts,Leen Bammens,Gilmer Govaert,Didac Carmona-Gutierrez,Frank Madeo,Karin Thevissen,Bruno P A Cammue

doi:10.3389/fmicb.2011.00047

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.

Highlights

Plant defensins are small, basic, cysteine-rich peptides that possess antifungal and in some cases antibacterial activity (Lay and Anderson, 2005; Aerts et al, 2008)
We determined the minimal inhibitory concentration (MIC) resulting in 100% growth inhibition of HsAFP1 for all individual yeast knock-out mutants and wild type (WT) yeast using twofold dilution
Blocking mitochondrial function by respiration inhibitor sodium azide antagonizes HsAFP1 antifungal capacity Several of the identified HsAFP1-tolerance or -sensitivity genes with a mitochondrial function are directly implicated in respiratory growth, i.e., RIP1, YDR115w, CEM1, ATP12, COX17, and MRPL35

Summary

Introduction

Basic, cysteine-rich peptides that possess antifungal and in some cases antibacterial activity (Lay and Anderson, 2005; Aerts et al, 2008). They are postulated to be part of the innate immune system of plants and protect them against invading fungal and bacterial pathogens. Plant defensins are nontoxic to human and plant cells (Thevissen et al, 2007a; Tavares et al, 2008), highlighting their therapeutic potential as novel antimycotics. Radish defensin RsAFP2 (Terras et al, 1992) was recently proven to be prophylactically effective against murine candidiasis, and this at least to the same extent as the antimycotic fluconazole (Tavares et al, 2008)

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