Tick defensin γ-core reduces Fusarium graminearum growth and abrogates mycotoxins production with high efficiency

Abstract

Fusarium graminearum is a major fungal pathogen affecting crops of worldwide importance. F. graminearum produces type B trichothecene mycotoxins (TCTB), which are not fully eliminated during food and feed processing. Therefore, the best way to minimize TCTB contamination is to develop prevention strategies. Herein we show that treatment with the reduced form of the γ-core of the tick defensin DefMT3, referred to as TickCore3 (TC3), decreases F. graminearum growth and abrogates TCTB production. The oxidized form of TC3 loses antifungal activity, but retains anti-mycotoxin activity. Molecular dynamics show that TC3 is recruited by specific membrane phospholipids in F. graminearum and that membrane binding of the oxidized form of TC3 is unstable. Capping each of the three cysteine residues of TC3 with methyl groups reduces its inhibitory efficacy. Substitutions of the positively-charged residues lysine (Lys) 6 or arginine 7 by threonine had the highest and the lesser impact, respectively, on the anti-mycotoxin activity of TC3. We conclude that the binding of linear TC3 to F. graminearum membrane phospholipids is required for the antifungal activity of the reduced peptide. Besides, Lys6 appears essential for the anti-mycotoxin activity of the reduced peptide. Our results provide foundation for developing novel and environment-friendly strategies for controlling F. graminearum.

Figure 1

Antifungal and anti-mycotoxin activity of natural and oxidized TC3. Effect of reduced TC3 (a–c) and oxidized TC3 (d–f) at 12.5, 25 and 50 µM on the fungal biomass weight of F. graminearum (a, d) and on the production of DON (b, e) and 15-ADON (c, f) in 10-day-old broths. Significant differences are labelled (*p < 0.05, **p < 0.01 and ***p < 0.005).

Figure 2

Membrane binding of TickCore3. The TC3 electrostatic potential of its residues are color-labeled as red (negative), grey (neutral) and blue (positive). The TC3 was placed 30 Å—from residue α-carbons to the phospholipid phosphorus atoms of the upper membrane surface. The upper and lower membrane leaflets, before and after 1 μs of MD, are shown with the majority of phospholipids depicted as sticks (grey = carbon, red = oxygen, and blue = nitrogen) (hydrogen atoms not shown). The main phospholipids (POPS, POPA and POPG) forming hydrogen bonds with TC3 are depicted as colored spheres (see legend for color scheme and percent composition). The line graph depicts the number hydrogen bond contacts (H-Bonds; y-axis) throughout the 1 μs of MD (x-axis; in ns). The dashed orange line in the graph is the average H-Bond contacts ($\overline{x}=9.7$) between the membrane and the TC3Ox.

Figure 3

Recruitment of TC3 by POPA and POPG phospholipids. The line graphs (A) are the distances measured (y-axis) between the α-carbon (αC) of TC3 residues and the phosphorus (P) atom of POPA (upper) and POPG (lower) throughout the 1 μs of MD (x-axis; in ns). The structural conformation at 0.939 μs of MD (B) shows the specific TC3 residues 4–6 that interact with POPA and POPG (hydrogen atoms not shown). The tertiary structure of TC3 also depicts the Cys residues (with hydrogen caps) and is color-coded from the amine-terminus (red) to the carboxyl-terminus (purple).

Figure 4

Antifungal and anti-mycotoxin activity of linear, methylated and oxidized TC3. Effect of reduced TC3, TC3-CH3-1, TC3-CH3-2, TC3-CH3-3, TC3-CH3-1 Ox, TC3-CH3-2 Ox, TC3-CH3-3 Ox and TC3-CH3-123 at 50 µM on the fungal biomass weight of F. graminearum (a) and on the production of DON (b) and 15-ADON (c) in 10-day-old broths. Significant differences are labelled (*p < 0.05, **p < 0.01 and ***p < 0.005).

Figure 5

Role of positively charged amino acids on the activity of TC3. Effect of reduced TC3, TC3-K6T, TC3-R7T, TC3-K13T or TC3-K14T at 12.5, 25 and 50 µM on the fungal biomass weight of F. graminearum (a) and on the production of DON (b) and 15-ADON (c) in 10-day-old broths. Significant differences are labelled (*p < 0.05, **p < 0.01 and ***p < 0.005).