| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1110-1 | ||||
Resumo:Disseminated and invasive diseases caused by opportunistic fungi have been recognized as a serious public health threat, mainly due to the increasing resistance to the few available drugs. Therefore, it is crucial to develop new therapeutic substances capable of treating these diseases. Among the candidates are the antimicrobial peptides (AMPs). Defensins, a prominent family of plant AMPs, exhibit strong antifungal activity and low toxicity to mammals, making them a promising source of new antifungal agents. Previously, we correlated the structure and biological activity of the γ-core motif of plant defensins, and based on this correlation we introduced specific modifications in certain positions and amino acids of the γ-core of Vigna unguiculata defensin (cowpea), increasing its charge and hydrophobicity, resulting in the peptides A36,42,44R37,38γ32-46VuDef (RR) and D-A36,42,44R37,38γ32-46VuDef (D-RR). RR and D-RR induced the death of Candida tropicalis at 6 and 3 h, with a lethal dose (DL100) of 27.5 and 23 µM, respectively. Both peptides showed no toxicity to mammalian cells and did not lyse ovine erythrocytes. We used several markers that indicated the cell death induced by RR was accidental and regulated, while for D-RR, it was a regulated cell death metacaspase independent. In both cases, there was a reduction in cell volume, indicating the role of K+, a regulator of fungal cell volume, in the cell death process. The aim of this study was to further investigate the mechanism of action of RR and D-RR-induced cell death in C. tropicalis, analyzing the interaction of the peptides and the involvement of ions. The role of K+ and Na+, low temperature (4°C), the protonophore CCCP, endocytosis inhibitors (nocodazole and latrunculin A), and the presence of competitors of polyamine channels (spermine and spermidine) were evaluated at DL100 and the time of cell death induced by the peptides. The effect of K+ addition at different times after peptide addition was also analyzed. The interaction dynamics of the peptides with the yeast were studied through 3D reconstruction and time-lapse by confocal with peptides labeled with 5-FAM (5-Carboxyfluorescein), and in the presence of the antioxidant ascorbic acid (AA) and K+. Our results showed partial protection of C. tropicalis cell death induced by the peptides at 4°C and by CCCP, indicating that the interaction of the peptides is energy-dependent. K+ and Na+ also provided protection from cell death, suggesting that the loss of these ions triggers a signal that leads to yeast death. The addition of K+ at different times after peptide addition showed a decrease in protection, indicating another signal, rather than K+ efflux, leading to cell death. Nocodazole and latrunculin A were not able to reverse the toxic effect of the peptides, ruling out endocytosis as the entry pathway for the peptides. Spermine partially protected cell death, indicating a role for polyamine transporters in the entrance of the peptides. The entry of peptides into the cytoplasm was confirmed by confocal, and it occurred even in the presence of AA and K+. These results indicate that RR and D-RR have an intracellular target, which needs to be confirmed by electron microscopy, and that the elimination of ROS by AA and the replenishment of K+ are the causes of protection, rather than the blocking of interaction by opposite charges neutralization between the peptides and the cell surface by the ions or AA. Palavras-chave: AMPs, antifungals, Candida tropicalis, cell death, mechanism of action Agência de fomento:CAPES, CNPq, FAPERJ | |||||