The antifungal peptide SmAPa1-21 derived from the g-core of defensin DefSm2-D induces cell-wall stress, membrane permeabilization, peroxisome biogenesis, oxidative stress and cell death in F. graminearum conidia. Role of His19 in peptide internalization

AGUSTINA FERNÁNDEZ; ISMAEL MALBRÁN; FANNY GUZMÁN; LAURA SUSANA BAKÁS; SANDRA ELIZABETH VAIRO CAVALLI

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Plant defensins are small cationic proteins ubiquitously expressed in the plant kingdom involved in innate immune defense. The primary structures are variable, but they all share the same tertiary structure, α/β structure stabilized by disulfide bridges: the so-called CSα/β fold. Two highly conserved regions have been identified in these proteins: the γ-core (GXCX3–9C), a well known determinant of the antimicrobial activity among disulphide-containing antimicrobial peptides and the α-core (GXCX3–5C), a less studied motif only present in plant defensins. The relevance 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 conidia from the phytopathogenic fungus Fusarium graminearum and we have characterized their action. Here, we continue the study of SmAP⍺1-21 mode of action (sequence: KLCEKPSKTWFGNCGNPRHCG; Minimum Inhibitory Concentration, MIC: 32 μM) and explore the correlation between the biological activity and primary structure of the α-core of DefSm2-D flower defensin focusing on the relevance of histidine 19. New peptides were designed by modifying the parent peptide (SmAPH19R and SmAPH19A, where His19 was replaced by Arg or Ala, respectively) and synthesized by the Fmoc solid phase method. Antifungal activity was determined against F. graminearum. Conidia membrane permeability was assessed by visualizing the influx of the membrane impermeant fluorescent red dye propidium iodide by confocal laser scanning microscopy (CLSM) after challenging conidia with each peptide. Reactive oxygen species (ROS) production was monitored on conidia with H2DCF-DA probe by fluorescence spectroscopy and CLSM. The peptides were derivatized with fluorescein and rhodamine B and subcellular localization in conidia was studied by CLSM by colocalization with the cell wall marker Trypan Blue. Transmission electron microscopy (TEM) was used to study the ultrastructural effects of SmAPα1-21 in conidial cells. SmAP⍺1-21 induced morphological changes in the cell wall and peroxisome biogenesis in F. graminearum conidia. SmAP2H19A and SmAP2H19R were found to be active against F. graminearum (MIC SmAPH19R: 40 µM and SmAPH19: 100 µM). The replacement of His19 by Ala produced a decrease in the net charge of one unit at pH 5.5 with a significant increase in MIC, thus evidencing the importance of the positive charge in position 19 of the antifungal peptide. All three peptides produced permeabilization of the conidia membrane and induced oxidative stress through ROS production. However, the replacement by Ala turned all the processes slower. SmAPH19R and SmAPH19A were localized in the conidia cell wall whereas SmAP⍺1-21 was internalized, first entering through the basal and apical cells of the macroconidia. As the incubation times were prolonged, SmAP⍺1-21 localized in all the cells of the spores with a non homogeneous distribution in the cell cytoplasm. SmAP⍺1-21 has a multi-step mechanism of action against F. graminearum conidia that involves at least alteration of the fungal cell wall, membrane permeabilization, peroxisome biogenesis, and induction of oxidative stress. The extracellular localization of peptides SmAPH19R and SmAPH19A highlights the role of the His 19 residue in the internalization.