Improved smallest peptides based on positive charge increase of the γ-core motif from Pν D1 and their mechanism of action against Candida species

Abstract

Background: Plant defensins have a hallmark γ-core motif (GXCX_3-9C) that is related to their antimicrobial properties. The aim of this work was to design synthetic peptides based on the region corresponding to the PvD₁ defensin γ-core that are the smallest amino acid sequences that bear the strongest biological activity.

Methods: We made rational substitutions of negatively charged amino acid residues with positively charged ones, and the reduction in length in the selected PvD₁ γ-core sequence to verify whether the increased net positive charges and shortened length are related to the increase in antifungal activity. Herein, we opted to evaluate the action mechanism of γ_33-41 PvD₁ ⁺⁺ peptide due to its significant inhibitory effect on tested yeasts. In addition, it is the smallest construct comprising only nine amino acid residues, giving it a better possibility to be a prototype for designing a new antifungal drug, with lower costs to the pharmaceutical industry while still maintaining the strongest antimicrobial properties.

Results: The γ_33-41 PvD₁ ⁺⁺ peptide caused the most toxic effects in the yeast Candida buinensis, leading to membrane permeabilization, viability loss, endogenous reactive oxygen species increase, the activation of metacaspase, and the loss of mitochondrial functionality, suggesting that this peptide triggers cell death via apoptosis.

Conclusion: We observed that the antifungal activity of PvD₁ is not strictly localized in the structural domain, which comprises the γ-core region and that the increase in the net positive charge is directly related to the increase in antifungal activity.

Keywords: antimicrobial peptide; cationic; defensin; mechanism of action.

Figure 1

Design, alignment and biochemical characteristics of the synthetic peptides. Notes: (A) Alignment of the primary structures of PνD₁ and the four synthetic peptides in amino acid one-letter code. Numbers above the PνD₁ sequence indicate the peptide size in amino acids. The amino acid residues in blue represent the PνD₁ γ-core region. The amino acid residues in red represent the replaced residues in the original PνD₁ sequence as follows: C residues were replaced by A, and D residues were replaced by R. Amino acid residues in black are not part of the γ-core region and were not changed. Numbers in the synthetic peptide names stand for the amino acid position in the original PνD₁. Double-plus (⁺⁺) indicates the double replacement from the original D to R amino acid residues to increase the synthetic peptide positive net charge. (B) Biochemical characteristics of the synthetic peptides.

Figure 2

Antifungal effects of _γ31-45PνD₁ and γ_31-45PνD₁⁺⁺ incubated for 24 hours at different concentrations on Candida albicans and Candida buinensis. *Indicates significance by the one-way analysis of variance (ANOVA) (P<0.05).

Figure 3

Antifungal effect of γ_33-41PνD₁ and γ_33-41PνD₁⁺⁺ incubated for 24 hours at different concentrations on Candida albicans and Candida buinensis. *Indicates significance by the one-way analysis of variance (ANOVA) (P<0.05).

Figure 4

MIC determination and cell viability loss. Notes: (A) Images of the plate wells at the end of the growth inhibition assay (at 24 hours) showing the growth pattern of Candida buinensis cells at the bottom of the wells in the absence (control) and in the presence of different γ_33-41PνD₁⁺⁺ concentrations. (B) Table showing the number of CFU and the percentage of viability loss of C. buinensis cells after treatment with 36.7 and 73.4 µM of γ_33-41PνD₁⁺⁺ for 24 hours, respectively. *Indicates significance by the one-way analysis of variance (ANOVA) (P<0.05). (C) Images of Petri dishes showing the CFU as described in (B). The experiments were carried out in triplicate. Abbreviations: CFU, colony forming units; MIC, minimal inhibitory concentration.

Figure 5

Images of membrane permeabilization assay of Candida buinensis cells after treatment with γ_33-41PνD₁⁺⁺ (25 µM) for 24 hours. Control cells were treated only with the Sytox green probe and positive control cells were treated with 300 mM ethanol. Bars =20 µm. Abbreviation: DIC, differential interference contrast.

Figure 6

Images of reactive oxygen species assay detection in Candida buinensis cells after treatment with γ_33-41PνD₁⁺⁺ (25 µM) for 24 hours. Control cells were treated only with the 2′,7′-dichlorofluoresceindiacetate probe and positive control cells were treated with 300 mM hydrogen peroxide. Bars =20 µm. Abbreviation: DIC, differential interference contrast.

Figure 7

Images of mitochondrial functionality assay of Candida buinensis cells after treatment with γ_33-41PνD₁⁺⁺ (25 µM) for 12 and 24 hours. Control cells were treated only with Rhodamine 123 probe and positive control cells were treated with 300 mM ethanol. Bars =20 µm. Abbreviation: DIC, differential interference contrast.

Figure 8

Images of detection of metacaspase activity assay of Candida buinensis cells after treatment with γ_33-41PνD₁⁺⁺ (25 µM) for 24 hours. Control cells and cells treated with γ_33-41PvD₁⁺⁺ were incubated with CaspACE FITC-VAD-FMK probe. Positive control cells were treated with 300 mM acetic acid and analyzed by fluorescence microscopy. Bars =20 µm. Abbreviation: DIC, differential interference contrast.