The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization.

Tanne L Cools,Guilherme D Brand,Liesbeth Demuyser,Barbara Koch,Jeroen Lammertyn,Carlos Bloch,Soňa Kucharíková,Kim Vriens,Sara Verbandt,Bruno P A Cammue,Caroline Struyfs,Ana Traven,Jan W Drijfhout,Dragana Spasic,Marcelo H S Ramada,Patrick Van Dijck,Karin Thevissen

doi:10.3389/fmicb.2017.02295

Abstract

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP’s). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP’s is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1’s internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.

Highlights

Plant defensins are peptides (45–54 amino acids), which are suggested to be part of the immune system of plants
The other positively charged amino acids in the C-terminal part of HsAFP1 (H40, lysine (K) 47 and K51), which seem not important for phosphatidic acid (PA) binding, are differently located. This is in line with reports of other proteins that bind to PA, such as mTOR, in which multiple amino acids contribute to PA docking, with an arginine being necessary for this interaction (Veverka et al, 2008)
We showed that HsAFP1 can bind to various lipids which contain at least one phosphate group, with PA being the lipid binding partner resulting in the most pronounced HsAFP1 binding

Summary

Introduction

Plant defensins are peptides (45–54 amino acids), which are suggested to be part of the immune system of plants. The antifungal mode of action of various plant defensins has been studied (as reviewed by Vriens et al, 2014) Plant defensins such as NaD1 (Poon et al, 2014), TPP3 (Baxter et al, 2015) and MtDef (Sagaram et al, 2013), isolated from tobacco, tomato and barrel clover, respectively, interact with fungal phospholipids, whereas others such as RsAFP2 (Thevissen et al, 2004), Psd (de Medeiros et al, 2010) and DmAMP1 (Thevissen et al, 2000, 2003), isolated from radish, pea and dahlia, respectively, interact with fungal-specific sphingolipids like glucosylceramides and mannosyl diinositolphosphoryl ceramides in the membranes of susceptible fungi. The latter is suggested to be a non-specific secondary effect rather than the primary cause of microbial killing (Thevissen et al, 1996)

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Conclusion