Antifungal and antibiofilm activity of the protease-resistant peptides NET1 and NET3 against Candida reference strains, with exploratory analysis of mitochondrial and membrane-associated changes

The global rise of drug-resistant fungal pathogens, particularly non- albicans Candida species, necessitates the development of novel therapeutic strategies. In this study, we investigated the antifungal, antibiofilm, and mitochondrial/membrane-disruptive activities of NET1 and NET3, two novel, protease-resistant synthetic antimicrobial peptides (AMPs) composed of D- and L-amino acid combinations, against reference strains of Candida albicans , Candida krusei ( Pichia kudriavzevii ), Candida lusitaniae ( Clavispora lusitaniae ), and Candida parapsilosis . Minimum inhibitory concentration (MIC) values for both peptides ranged from 8 to 16 μg/mL across all tested strains. Notably, both peptides inhibited initial biofilm formation at sub-MIC levels in C. albicans (MBICs of 2 μg/mL for NET1 and 1 μg/mL for NET3; 8- to 16-fold below their MICs). To elucidate the cellular mechanisms accompanying fungal growth inhibition, key mitochondrial and membrane-associated changes and membrane hallmarks were evaluated following peptide exposure. At 0.5 × MIC, JC-1 fluorescent staining revealed that both NET1 and NET3 induced widespread collapse of the mitochondrial membrane potential. At 1 × MIC, subcellular fractionation and spectrophotometric analysis at 550 nm showed a marked shift of Cytochrome c from the mitochondrial to the cytosolic fraction, consistent with outer mitochondrial membrane permeabilization. Furthermore, CF®488A Annexin V and Propidium Iodide (PI) dual-staining at 0.5 × MIC revealed phosphatidylserine externalization with intact membranes (predominantly early apoptotic-like Annexin V + /PI − cells) in C. albicans and C. parapsilosis , and dual-positive staining (Annexin V + /PI + , consistent with late apoptosis-like progression or secondary membrane permeabilization) in peptide-treated C. krusei and C. lusitaniae ; primary necrotic (Annexin V − /PI + ) cells were rarely detected at sub-MIC levels. These results suggest that the antifungal activity of NET1 and NET3 involves a mitochondria-associated, apoptosis-like response alongside membrane disruption rather than primary necrosis alone. Overall, these protease-resistant AMPs represent promising therapeutic candidates for combating opportunistic Candida infections.