Improved smallest peptides based on positive charge increase of the &gamma;-core motif from <em>P&nu;</em>D₁ and their mechanism of action against <em>Candida</em> species

Abstract

BackgroundPlant defensins have a hallmark γ-core motif (GXCX3-9C) 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.MethodsWe 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 γ33-41PvD1++ 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.ResultsThe γ33-41PvD1++ 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.ConclusionWe 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.

Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi

“…A peptide belonging to the thionin family, viscotoxin A3 (VtA3), has also been shown to be capable of altering the permeability of the plasma membrane, and it has also been observed that this peptide is internalized by F. solani cells [39]. Many works report that after membrane interaction, AMPs can trigger a series of subsequent events such as the induction of reactive oxygen species (ROS), the inhibition of protein synthesis, the inhibition of mitochondrial activity and the triggering signaling cascades leading to apoptosis [40,41]. Taveira et al [42] showed CaThi has strong antimicrobial activity against six Candida species and caused plasma membrane permeabilization in all tested yeasts.…”

“…An HsAFP1 a defensin from Heuchera sanguinea was also able to induce intracellular accumulation of ROS, and activated caspases were required to cause apoptosis in C. albicans cells [48]. A synthetic plant defensin (γ 33 -41 PvD 1 ++ ) was also able to activate metacaspases in C. buinensis cells and yet cause a collapse of the mitochondrial membrane potential in these cells [41]. Taveira et al [49].…”

Plant Defensins from a Structural Perspective

“…Several structure-activity studies indicate that the major determinants of the antifungal and antibacterial activity of plant defensins reside in the γ-core motif [ 42 , 54 , 79 , 89 , 99 ]. Furthermore, the γ-core motif peptides derived from defensins RsAFP-2, MtDef4, MtDef5, MsDef1, So-D2, Vu-Def from Vigna unguiculata , BcDef from Brugmansia x candida , PνD1 from P. vulgaris , and the tomato defensin SolyC07g007760 demonstrated antifungal and antibacterial activity at micromolar concentrations [ 21 , 42 , 99 , 100 , 101 , 102 , 103 , 104 ]. Additionally, specific regions within γ-core motifs of MsDef1 and MtDef4 defensins contribute to their different modes of antifungal action [ 105 ].…”

“…The main physicochemical features responsible for the functional importance of the γ-core motif include the net positive charge and the periodic placement of charged and hydrophobic amino acid residues [ 42 ]. Increasing the positive charge of the PνD1 defensin’s γ-core motif derived peptide by replacing two aspartic acids D37 and D38 with two arginines significantly enhanced its antifungal activity [ 99 ]. Many charged residues are located in the L3 loop of the γ-core motif connecting beta-strands β2 and β3.…”

“…Although some authors include only the GXCX3-9C sequence in the γ-core motif (hence, selecting shorter versions of γ-core motif for functional studies in some cases), based on the consensus between all disulfide-containing antimicrobial peptides [97,98], the γ-core motif includes full-length β2 and β3 strands and the loop L3 between them (Figure 6). Several structure-activity studies indicate that the major determinants of the antifungal and antibacterial activity of plant defensins reside in the γ-core motif [42,54,79,89,99]. Furthermore, the γ-core motif peptides derived from defensins RsAFP-2, MtDef4, MtDef5, MsDef1, So-D2, Vu-Def from Vigna unguiculata, BcDef from Brugmansia x candida, PνD1 from P. vulgaris, and the tomato defensin SolyC07g007760 demonstrated antifungal and antibacterial activity at micromolar concentrations [21,42,[99][100][101][102][103][104].…”