The antifungal plant defensin HsAFP1 is a phosphatidic acid-interacting peptide inducing membrane permeabilization

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

Research output: Contribution to journalArticleResearchpeer-review

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.

Original languageEnglish
Article number2295
JournalFrontiers in Microbiology
Volume8
Issue numberNOV
DOIs
Publication statusPublished - 21 Nov 2017

Keywords

  • Antifungal mode of action
  • Lipid membrane target
  • Membrane permeabilization
  • Peptide internalization
  • Plant defensins
  • Yeast

Cite this

Cools, T. L., Vriens, K., Struyfs, C., Verbandt, S., Ramada, M. H. S., Brand, G. D., ... Thevissen, K. (2017). The antifungal plant defensin HsAFP1 is a phosphatidic acid-interacting peptide inducing membrane permeabilization. Frontiers in Microbiology, 8(NOV), [2295]. https://doi.org/10.3389/fmicb.2017.02295
Cools, Tanne L. ; Vriens, Kim ; Struyfs, Caroline ; Verbandt, Sara ; Ramada, Marcelo H.S. ; Brand, Guilherme D. ; Bloch, Carlos ; Koch, Barbara ; Traven, Ana ; Drijfhout, Jan W. ; Demuyser, Liesbeth ; Kucharíková, Sona ; Van Dijck, Patrick ; Spasic, Dragana ; Lammertyn, Jeroen ; Cammue, Bruno P.A. ; Thevissen, Karin. / The antifungal plant defensin HsAFP1 is a phosphatidic acid-interacting peptide inducing membrane permeabilization. In: Frontiers in Microbiology. 2017 ; Vol. 8, No. NOV.
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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.",
keywords = "Antifungal mode of action, Lipid membrane target, Membrane permeabilization, Peptide internalization, Plant defensins, Yeast",
author = "Cools, {Tanne L.} and Kim Vriens and Caroline Struyfs and Sara Verbandt and Ramada, {Marcelo H.S.} and Brand, {Guilherme D.} and Carlos Bloch and Barbara Koch and Ana Traven and Drijfhout, {Jan W.} and Liesbeth Demuyser and Sona Kuchar{\'i}kov{\'a} and {Van Dijck}, Patrick and Dragana Spasic and Jeroen Lammertyn and Cammue, {Bruno P.A.} and Karin Thevissen",
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Cools, TL, Vriens, K, Struyfs, C, Verbandt, S, Ramada, MHS, Brand, GD, Bloch, C, Koch, B, Traven, A, Drijfhout, JW, Demuyser, L, Kucharíková, S, Van Dijck, P, Spasic, D, Lammertyn, J, Cammue, BPA & Thevissen, K 2017, 'The antifungal plant defensin HsAFP1 is a phosphatidic acid-interacting peptide inducing membrane permeabilization' Frontiers in Microbiology, vol. 8, no. NOV, 2295. https://doi.org/10.3389/fmicb.2017.02295

The antifungal plant defensin HsAFP1 is a phosphatidic acid-interacting peptide inducing membrane permeabilization. / Cools, Tanne L.; Vriens, Kim; Struyfs, Caroline; Verbandt, Sara; Ramada, Marcelo H.S.; Brand, Guilherme D.; Bloch, Carlos; Koch, Barbara; Traven, Ana; Drijfhout, Jan W.; Demuyser, Liesbeth; Kucharíková, Sona; Van Dijck, Patrick; Spasic, Dragana; Lammertyn, Jeroen; Cammue, Bruno P.A.; Thevissen, Karin.

In: Frontiers in Microbiology, Vol. 8, No. NOV, 2295, 21.11.2017.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The antifungal plant defensin HsAFP1 is a phosphatidic acid-interacting peptide inducing membrane permeabilization

AU - Cools, Tanne L.

AU - Vriens, Kim

AU - Struyfs, Caroline

AU - Verbandt, Sara

AU - Ramada, Marcelo H.S.

AU - Brand, Guilherme D.

AU - Bloch, Carlos

AU - Koch, Barbara

AU - Traven, Ana

AU - Drijfhout, Jan W.

AU - Demuyser, Liesbeth

AU - Kucharíková, Sona

AU - Van Dijck, Patrick

AU - Spasic, Dragana

AU - Lammertyn, Jeroen

AU - Cammue, Bruno P.A.

AU - Thevissen, Karin

PY - 2017/11/21

Y1 - 2017/11/21

N2 - 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.

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KW - Antifungal mode of action

KW - Lipid membrane target

KW - Membrane permeabilization

KW - Peptide internalization

KW - Plant defensins

KW - Yeast

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U2 - 10.3389/fmicb.2017.02295

DO - 10.3389/fmicb.2017.02295

M3 - Article

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JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

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