Role of calcium in reactive oxygen species-induced apoptosis in <i>Candida albicans</i>: an antifungal mechanism of antimicrobial peptide, PMAP-23

PMAP-23 (RIIDLLWRVRRPQKPKFVTVWVR-NH2) is an antimicrobial peptide (AMP) derived from porcine myeloid. Membrane disruption is thought to underpin the anticandidal activity of PMAP-23. However, many AMPs act via mechanisms other than simple membrane permeabilisation. Here, we investigated the anticandidal mechanism of PMAP-23 at low concentrations. Membrane disruption and depolarisation and rapid K+ efflux were observed in Candida albicans cells treated with 5 µM PMAP-23. In contrast, 2.5 µM PMAP-23 caused membrane depolarisation and K+ efflux without membrane disruption. The lower PMAP-23 concentration increased cytosolic and mitochondrial Ca2+ levels. Disruption of Ca2+ homeostasis altered the NAD+/NADH ratio and resulted in reactive oxygen species (ROS) accumulation and glutathione oxidation. PMAP-23 treatment also stimulated apoptosis, as evidenced by metacaspase activation, DNA fragmentation, and phosphatidylserine externalisation. Pretreatment with the mitochondrial Ca2+ uptake inhibitor (ruthenium red) or ROS scavenger (N-acetylcysteine) attenuated these apoptotic events. Our results suggest that PMAP-23 induces apoptosis as antifungal mechanism, and mitochondrial Ca2+-induced ROS is major factor to trigger the apoptosis. Thus, the anticandidal activity of PMAP-23 is not based solely on disruption of biological membranes but also involves induction of apoptosis via mitochondrial Ca2+-dependent ROS. PMAP-23 mode of action sheds new light on the antifungal mechanism of antimicrobial peptides, supporting the role of Ca2+ and ROS in apoptosis regulation.