Interaction Between the Plant ApDef1 Defensin and Saccharomyces cerevisiae Results in Yeast Death Through a Cell Cycle- and Caspase-Dependent Process Occurring Via Oxidative Stress

SOARES, JÚLIA RIBEIRO; TAVEIRA GB; JOSÉ TENÓRIO DE MELO, EDÉSIO; DE LA CANAL L; REGENTE M; GOMES, VALDIRENE MOREIRA; DA CUNHA, MAURA; PINEDO, M; CARVALHO, A. O.

Joinville, Santa Catarina, Brasil

Congreso; 47a Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular; 2018

Sociedade Brasileira de Bioquímica e Biologia Molecular

Introduction: Plant defensins were discovered at beginning of the 90s´ and present multiple biological activities, highlighting their antimicrobial activity. However, their precise mechanism of action is still unknown. ApDef1 is a defensin from seeds of Adenanthera pavonina which has fungicide activity on cells of S. cerevisiae, presenting a MIC of 7.8 µM and require a maximum of 18 h to cause the death of all cells used in the assay, under the conditions tested. Objectives: Herein, we studied ApDef1-Saccharomyces cerevisiae interaction and part of the mechanism involved. Material and Methods: ApDef1-S. cerevisiae interaction was studied through viability assays performed with hydroxyurea synchronized-yeast and pretreated with CCCP (Carbonyl cyanide 3-chlorophenylhydrazone). Yeast cells after treatment with ApDef1 were analyzed to verify the occurrence of plasma membrane permeabilization, ROS induction, chromatin condensation, and caspase activation which analyses were assessed through Sytox green, DAB (3,3′-diaminobenzidine), DAPI (4′,6-diamidino-2-phenylindole) and FITC-VAD-FMK (fluorescein isothiocyanate -alanyl-aspartyl-[O-methyl]-fluoromethylketone) probes, respectively. The involvement of oxidative stress and caspases in cell death induced by ApDef1 was verified through viability assay performed in presence of ascorbic acid and Z-VAD-FMK (carbobenzoxyvalyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) caspase inhibitor. The additional ultrastructural analysis was done by electron microscopy. Results and Discussion: After 3 h of treatment with ApDef1, 98.76% of synchronized cell population died. Pretreatment with CCCP protected yeast from ApDef1 induced death. ApDef1-S. cerevisiae interaction resulted in membrane permeabilization, H2O2 increased production, chromatin condensation and caspase activation. Ascorbic acid prevented yeast cell death and membrane permeabilization as well as the Z-VAD-FMK caspase inhibitor prevented yeast cell death. Conclusions: ApDef1-S. cerevisiae interaction caused cell death through cell cycle dependente process which requires preserved membrane potential. After interaction, yeast went through uncontrolled ROS production and accumulation, which led to plasma membrane permeabilization, chromatin condensation and, ultimately, cell death by activation of caspase-dependent apoptosis via.Keywords: Antimicrobial peptides, ROS induction, Cell cycle Support: CNPq, FAPERJ, CAPES.