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

FERNÁNDEZ, AGUSTINA; GONZÁLEZ, MARIANO; MALBRÁN, ISMAEL; MATÉ, SABINA; VAZQUEZ, ROMINA; GUZMÁN QUIMBAYO, FANNY; BAKÁS, LAURA; VAIRO CAVALLI, SANDRA

Congreso; XLIX Reunión Anual SAB; 2021

Sociedad Argentina de Biofísica (SAB)

Plant defensins are antimicrobial peptides (AMPs); they are small basic, cysteine-richpeptides ubiquitously expressed in the plant kingdom and mostly involved in hostdefence. They present a highly variable sequence but a conserved structure. Two highlyconserved regions have been identified in these proteins: the γ-core (GXCX3?9C), a well-known determinant of the antimicrobial activity among disulphide-containing AMPs andthe α-core (GXCX3?5C), a less studied and not conserved motif among them. Theimportance of these motifs relies on the presence of positive residues which would allowthe interaction with negative charges on the pathogen membrane and/or cell wall. Inprevious studies, we have demonstrated that synthetic peptides derived from theseregions are active at micromolar concentrations against the conidia from thephytopathogenic fungus Fusarium graminearum and we have characterized their action.In order to gain insight into the understanding of the α-core, new peptides were designedby modifying the parent peptide SmAPa1-21 (Sequence: KLCEKPSKTWFGNCGNPRHCG;Minimum Inhibitory Concentration MIC: 32 μM), which caused membranepermeabilization, induced morphological changes in the cell wall and the interaction withPOPC/Erg monolayers (3:1) revealed its monolayer insertion. Three peptides weresynthesized: SmAPH19R, where His19 was modified by Arg; SmAPH19A, in which His19was modified by Ala and cSmAPC14S, a cyclic peptide synthesized promoting anintramolecular disulfide bond and Cys14 was modified by Ser. The new peptides werefound to be active against F. graminearum (MIC SmAPH19R: 40 µM, SmAPH19: 100 µMand cSmAPC14S: 70 µM). Both SmAPH19R and cSmAPC14S produced permeabilizationof the conidia membrane, while SmAPH19A did not. All three peptides induced oxidativestress in the treated conidia through ROS production, as assessed by confocal microscopyand fluorometric measurements. SmAPH19R caused conidia death in 3 h, SmAPH19A in 6h and cSmAPC14S in 30 min. Confocal microscopy of the peptides derivatized withfluorescein showed that SmAPH19R and SmAPH19A were localized in the conidia cellwall. The change of His by Ala reduced the activity compared with the parent peptide andwould produce a change in the mechanism of action, since membrane permeabilizationdid not occur under the assay conditions. Although cSmAPC14S exhibited a higher MICthan the parent peptide, it was the most lethal on the time to kill assay.