A role in intracellular K+ in protecting pathogenic dimorphic fungi against induced cell death by bioinspired antimicrobial peptides

• Published in: Biochimica et biophysica acta. General subjects Vol. 1869; no. 6; p. 130795
• Main Authors: Damica, Filipe Zaniratti, Lucas, Douglas Ribeiro, Toledo, Estefany Bras, de Carvalho Ribeiro, Marilúcia, Façanha, Anna Lvovna Okorokova, Zeraik, Ana Eliza, Seabra, Sérgio Henrique, da Silva, Juliana Azevedo, Gomes, Valdirene Moreira, de Oliveira Carvalho, André
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2025
• Subjects: Antifungal Agents - chemistry; Antifungal Agents - pharmacology; Antimicrobial Peptides - chemistry; Antimicrobial Peptides - pharmacology; Candida albicans - drug effects; Candida albicans - metabolism; Candida tropicalis - drug effects; Candida tropicalis - metabolism; Cell Death - drug effects; Extracellular acidification; Internalization of peptides; Ion flux, Ultrastructure; Mitochondria - drug effects; Mitochondria - metabolism; Oxidative Stress - drug effects; Potassium - metabolism; AA; RR; ΔpH; SSC; AMP; Internalization of peptides; H2DCFDA; dAMP; 4-AP; NPPB; TEA; CFU; Δψ; D-RR; FSC; ROS; Extracellular acidification; PI; 5-FAM; WR; Ion flux, Ultrastructure; AcA; DIC
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2025.130795

Antimicrobial peptides (AMPs) are promising drugs, though their fungal combat mechanisms remain partly unclear. We designed three AMPs (dAMPs) based on the γ-core of the Vu-Def1 seed defensin from Vigna unguiculata L. Walp. named RR, D-RR, and WR, and assessed their actions on Candida tropicalis and Candida albicans. Amidst their actions are cell shrinkage caused by K+ efflux from fungal cells. K+ involvement in fungal death by these peptides was explored. We assessed cell shrinkage, oxidative stress, mitochondria hyperpolarization, membrane permeabilization, medium acidification, antimicrobial activity under hypoosmotic conditions, and cellular degradation. Viability assays were performed with channel blockers and K+ addition at various times. The interactions of dAMPs with salts and fungal cells were analyzed using circular dichroism and microscopy. K+ and Cl− channels were not directly involved in dAMPs-induced death. Supplementation with K+ protected fungal cells from death. In tests, cations often deactivated them through charge neutralization. Peptides maintained their conformation with K+ and were found in cell cytoplasm indicating K+ did not neutralize charges. K+ did not prevent oxidative stress, but protected from cell shrinkage and mitochondria hyperpolarization. dAMPs rapidly stimulated medium acidification, followed by inhibition after 1 min, and K+ prevented acidification. Membrane permeabilization occurred after 20 min, faster with WR, explaining lack of protection from blockers. Fungal death was accelerated under hypoosmotic conditions. Electrophoresis revealed protein degradation, while ultrastructural analysis of the cells showed vacuolization, indicative of cytoplasmic degradation. Thus, K+ prevented cell death by maintaining internal levels, averting activation of cell degradation process. •The peptides enter the fungal cytoplasm, and the entrance is not precluded by K+.•K+ did not block the toxicity of the peptides by surface charge neutralization.•K+ addition to the medium protects fungi from death induced by the peptides.•Fungi preincubated under hypoosmotic conditions were killed faster by the peptides.•K+ protection is related to the maintenance of its critical intracellular concentration.