Peptides derived from the alpha-core of a Silybum marianum defensin induced membrane permeabilization, oxidative stress and cell death in Fusarium graminearum conidia

AGUSTINA FERNÁNDEZ; MARIANO GONZÁLEZ; ISMAEL MALBRÁN; SABINA MATÉ; ROMINA VÁZQUEZ; FANNY GUZMÁN; LAURA SUSANA BAKÁS; SANDRA ELIZABETH VAIRO CAVALLI

Congreso; XLIX Reunión Anual de la Sociedad Argentina de Biofísica; 2021

Sociedad Argentina de Biofísica

Plant defensins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. Two highly conserved regions have been identified in these proteins: the β-core (GXCX3-9C), a well-known determinant of the antimicrobial activity among disulphide-containing AMPs and the α-core (GXCX3-5C), a less studied and not conserved motif among them. The importance of these motifs relies on the presence of positive residues which would allow the interaction with negative charges on the pathogen membrane and/or cell wall. In previous studies, we have demonstrated that synthetic peptides derived from these regions are active at micromolar concentrations against the conidia from the phytopathogenic fungus Fusarium graminearum and we have characterized their action. In order to gain insight into the understanding of the α-core, new peptides were designed by modifying the parent peptide SmAPa1-21 (Sequence: KLCEKPSKTWFGNCGNPRHCG; Minimum Inhibitory Concentration MIC: 32 µM), which caused membrane permeabilization, induced morphological changes in the cell wall and the interaction with POPC/Erg monolayers (3:1) revealed its monolayer insertion. Three peptides were synthesized: SmAPH19R, where His19 was modified by Arg; SmAPH19A, in which His19 was modified by Ala and cSmAPC14S, a cyclic peptide synthesized promoting an intramolecular disulfide bond and Cys14 was modified by Ser. The new peptides were found to be active against F. graminearum (MIC SmAPH19R: 40 µM, SmAPH19: 100 µM and cSmAPC14S: 70 µM). Both SmAPH19R and cSmAPC14S produced permeabilization of the conidia membrane, while SmAPH19A did not. All three peptides induced oxidative stress in the treated conidia through ROS production, as assessed by confocal microscopy and fluorometric measurements. SmAPH19R caused conidia death in 3 h, SmAPH19A in 6 h and cSmAPC14S in 30 min. Confocal microscopy of the peptides derivatized with fluorescein showed that SmAPH19R and SmAPH19A were localized in the conidia cell wall. The change of His by Ala reduced the activity compared with the parent peptide and would produce a change in the mechanism of action, since membrane permeabilization did not occur under the assay conditions. Although cSmAPC14S exhibited a higher MIC than the parent peptide, it was the most lethal on the time to kill assay.