| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1248-1 | ||||
Resumo:Recently the World Health Organization (WHO) issued the “fungal priority pathogens list to guide research, development and public health action” which is the first global effort to systematically prioritize fungal pathogens, the report presents categories and classifies four fungi as “priority group critical” among these two species of the genus Candida were classified as, C. albicans and C. auris. This report was issued to strengthen the global response to fungal infections and antifungal resistance by highlighting the need for the development of new therapeutic substances. In view of this, antimicrobial peptides (AMPs) are defense molecules of organisms and have extensive inhibitory activity against microorganisms, thus being a promising class of new therapeutic substances. Our group has been working with an AMP named Vu-Def1, from the defensin family, isolated from Vigna unguiculata (cowpea). From the γ-core of Vu-Def1 and a study where we correlated the primary structure of the γ-cores and the biological activity of several defensins, we designed the peptide A42,44R37,38W36-39γ32-46Vu-Def (WR), more cationic and hydrophobic than the original, in order to improve its biological activity. Previously, WR demonstrated strong antifungal activity against Candida yeasts, and low toxicity to human monocytes and murine macrophages. For C. albicans, a lethal dose of 27.5µM and a time of death of 1h were determined. We verified by several probes that WR affects intracellular organelles, with emphasis on vacuolar and mitochondrial dysfunction that induces accidental cell death in C. albicans. However, we observed cell shrinkage that is not associated with this type of death. Due to the dysfunction observed in intracellular organelles and cell shrinkage, the objective of this work was to study the interaction and entry of WR in C. albicans and we also analyzed the role of the K+ ion, described as a regulator of cell volume in fungi. For this, we performed an assay with 100 and 200mM of K+ and Na+ phosphates, CaCl2 and 5mM EGTA, where we observed that all ions partially reversed death. To determine whether WR enters the cell, we performed the assay under a confocal microscope with Calcofluor White 2% and WR labeled with the 5-FAM fluorophore and concluded that there was internalization of WR, and that entry occurred even in the presence of K+, indicating that WR previously observed protection may not be related to charge blocking between WR and the cell surface. To differentiate the effect of interaction with membranes, we tested the hemolytic activity of WR and there was no erythrocyte lysis, and using Circular Dichroism we analyzed the biophysical interactions of WR with models of artificial membranes (SUVs). We observed that in water or in K+ or Na+ phosphate and in the presence of POPC, POPC/POPE or ergosterol 15 and 35% the WR conformation is disordered and only in TFE (100%) and POPC/POPG, there is the formation of α -propellers. Our results suggest that WR does not target the plasma membrane and the ions do not block charge, but instead replace what the cell is losing, blocking the signal that leads to death. Palavras-chave: Antimicrobial peptides, Mechanism of interaction, Mechanism of Action, , fungal diseases, role of the ions Agência de fomento:Universidade Estadual do Norte Fluminense “Darcy Ribeiro” / Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro - FAPERJ. | |||||