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

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.

FIGURE 1

Lipid interaction partners of HsAFP1 determined via (A,B) protein-lipid overlay assay using PIP Strips, (C) rELISA and (D) phospholipid vesicles. (A) The PIP Strips were incubated with HsAFP1 (lower membrane) or without HsAFP1 (top control membrane) and subsequently with HsAFP1 antiserum, anti-rabbit IgG-Alkaline Phosphatase and 4-nitrophenyl phosphate. Colored spots represent HsAFP1 binding events, as indicated by black arrows (A), which were quantified via Image Studio Lite (B). (C) Optical density (OD_{405 nm}) values representing the interaction of 12.5 μM HsAFP1 with the control (MeOH) and all tested phospholipids. Data are means ± SEM, for n ≥ 3 experiments. Significant differences between HsAFP1-MeOH and HsAFP1-lipid interactions were determined via one-way ANOVA followed by Dunnett multiple comparison, with ^∗∗∗ and ^∗∗∗∗ representing P < 0.001 and P < 0.0001, respectively. (D) Thermograms of phosphatidylcholine(PC)/phosphatidylglycerol(PG) (75/25) and PC/PG/phosphatidic acid(PA) (75/15/10) vesicles in absence or presence of HsAFP1, as determined via DSC experiments. Representative graphs of the phase transition of 0.5 mM liposomes in absence or presence of 40 μM HsAFP1 are presented in black, with the corresponding fit (using a non-two state model) shown in red. Both the fit of the two peaks as well as the overall fit were presented in red. Phase transition temperatures of both peaks of the fit (Tm1 and Tm2) ± SD are specified in the box.

FIGURE 2

Amino acid sequence alignment of HsAFP1 with HsAFP1 mutant HsAFP1[H32A][R52A], HsAFP1-derived peptide fragments (HsLin01-HsLin06), HsLin06-derived peptide fragments (HsLin06_02;05;09;01;03;06;10 and 15) and the plant defensins NsD7, MtDef4 and NaD2. Gray bars indicate the highly conserved amino acids among plant defensins. The blue, green, and the orange boxes represent the position of the PA binding domain, the amino acid substitutions and the γ-core, respectively. (-) denote gaps in the alignment.

FIGURE 3

Amino acids of HsAFP1 important for phosphatidic acid (PA) binding, using competitive rELISA assays. Competition of 12.5 μM HsAFP1 with 50 μM HsLin01-06 (A), C-truncated HsLin06 variants (HsLin06_02;05;09 in Figure 2; (B) or N-truncated variants (HsLin06_01;03;06;10;15 in Figure 2; (C) with both peptides co-incubated. Data are means ± SEM, for n ≥ 3 experiments. Data are expressed relative to the HsAFP1-PA binding without HsLin. Significant differences between HsAFP1 and HsAFP1 + 4x HsLin interactions were determined via one-way ANOVA followed by Dunnett multiple comparison, with ^∗, ^∗∗∗, and ^∗∗∗∗ representing P < 0.05, P < 0.001, and P < 0.0001, respectively. (D) 3D structure of HsAFP1 with the positively charged amino acids [histidine (H) 32 and 40, lysine (K) 47, K51 and arginine (R) 52] of HsLin06 drawn in blue on its backbone, using the Chimera visualization program. Green clustered amino acids (H32 and R52) are important for PA binding while the red clustered (H40, K47 and K51) are not.

FIGURE 4

Reduced phospholipid binding capacity (A) and antifungal activity (B) of HsAFP1[H32A][R52A] compared to native HsAFP1. Data are means ± SEM, for n ≥ 3 experiments. (A) Optical density (OD_{405 nm}) values representing the interaction of 12.5 μM HsAFP1 or HsAFP1[H32A][R52A] with all phospholipids tested [phosphatidic acid (PA), phosphatidylinositol(3,5)bisphosphate (PtdIns(3,5)P₂), phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P₂)], determined via rELISA assays. Significant differences between HsAFP1 and HsAFP1[H32A][R52A] were determined via two-way ANOVA followed by Sidak multiple comparison, with ^∗∗∗ and ^∗∗∗∗ representing P < 0.001 and P < 0.0001, respectively. (B) Minimum Inhibitory Concentration of HsAFP1 or HsAFP1[H32A][R52A] resulting in 50% cell growth reduction (MIC50) of F. culmorum and Minimum Fungicidal Concentration of HsAFP1 or HsAFP1[H32A][R52A] resulting in 50% cell death (MFC50) of S. cerevisiae. Significant differences between HsAFP1 and HsAFP1[H32A][R52A] were determined via an unpaired student t-test with Welch’s correction, with ^∗ representing P < 0.05.

FIGURE 5

Phosphate-dependent binding of HsAFP1 to phosphatidic acid (PA), determined via rELISA assays. Interaction of 12.5 μM HsAFP1 with PA or methyl-PA (A) or for the interaction of HsAFP1 with PA in buffers with different phosphate concentrations (B). Data are means ± SEM, for n ≥ 3 experiments. Data are expressed relative to the HsAFP1-PA binding (A) or relative to the HsAFP1-PA binding in demi-water (0 M NaH₂PO₄) (dotted line) (B). Significant differences between PA and methyl-PA interactions were determined via an unpaired student t-test with Welch’s correction (A), while significant differences between demi-water (0M NaH₂PO₄) and phosphate buffers were determined via one-way ANOVA followed by Dunnett multiple comparison (B), with ^∗∗ and ^∗∗∗ representing P < 0.01 and P < 0.001, respectively.

FIGURE 6

HsAFP1 localization in S. cerevisiae cells treated with high (48 μM) BODIPY-HsAFP1 concentrations. Confocal microscope images of 150 min-treated S. cerevisiae cultures with 48 μM BODIPY-HsAFP1. Propidium iodide (PI; 2 μg/mL) was used to identify membrane permeabilization. (A) Confocal images acquired with normal laser intensities [Alexa fluor 488-10% (argon laser)-HV451 and PI-1% (559nm laser)-HV405]. (B) Confocal images taken at high laser intensities [Alexa fluor 488-10% (argon laser)-HV573 and PI-1% (559 nm laser)-HV566], without impacting autofluorescence. Representative BODIPY-HsAFP1+/PI- cells are indicated with arrows. Bar: 5 μm.

FIGURE 7

BODIPY-HsAFP1 is located intracellularly in membrane-permeabilized cells and at the cell surface of non-membrane permeabilized S. cerevisiae cells treated with 16 μM BODIPY-HsAFP1. Confocal microscope images of 60 min treated S. cerevisiae cultures with 16 μM BODIPY-HsAFP1. Propidium iodide (PI; 2 μg/mL) was used to identify membrane permeabilization. (A) Confocal images acquired with normal laser intensities (Alexa fluor 488-10% (argon laser)-HV573 and PI-1% (559 nm laser)-HV600). (B) Confocal images taken at high laser intensities [Alexa fluor 488-10% (argon laser)-HV700 and PI-1% (559 nm laser)-HV600], without impacting autofluorescence. Representative BODIPY-HsAFP1 accumulation spots at the cell surface are indicated with arrows. Bar: 5 μm.

FIGURE 8

Kinetics of HsAFP1 internalization and membrane permeabilization in S. cerevisiae treated with (A) low (4 μM) or (B) high (48 μM) BODIPY-HsAFP1 (B-Hs) and 2 μg/mL propidium iodide (PI), determined via flow cytometry. The gray dashed line represents the percentage of B-Hs+ yeast cells in the population, relative to control (MQ water) treatment, which can be further divided in two subpopulations: yeast cells with (PI+; orange squares) and without (PI–; black circles) compromised membranes. Data are means ± SEM, for n = 3 experiments. To analyze significant differences in the size of the subpopulations between the time zero and other time points, one-way ANOVA followed by Dunnett multiple comparison was performed.