Calcium homeostasis plays important roles in the internalisation and activities of the small synthetic antifungal peptide PAF26

Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here we investigate the mode-of-action of the small, rationally designed synthetic antifungal peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell killing activity of PAF26 is dependent on extracellular Ca2+ and the presence of fully functioning fungal Ca2+ homeostatic/signalling machinery. In a screen of mutants with deletions in Ca2+-signalling machinery, we identified three mutants more tolerant to PAF26. The Ca2+ ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the cell envelope. The vacuolar Ca2+ channel YVC-1 was shown to be essential for its accumulation and concentration within the vacuolar system. The Ca2+ channel CCH-1 was found to be required to prevent the translocation of PAF26 across the plasma membrane. In the wild type, Ca2+ removal from the medium resulted in the peptide remaining trapped in small vesicles as in the{Delta} yvc-1 mutant. It is therefore apparent that cell killing by PAF26 is complex and unusually dependent on extracellular Ca2+ and components of the Ca2+-regulatory machinery. AUTHOR SUMMARYLife threatening diseases can be caused when fungi invade human tissues. These invasions often occur when a persons immune defences are down, often due to treatments for cancer or transplantation. These infections are commonly buried deep within the body and as such are difficult to access and treat. Current medications are often highly toxic to the patient. There is also a worrying rise in drug resistance seen in fungi sampled from patients, with infections effectively untreatable - a death sentence. Antifungal peptides such as PAF26 provide a possible solution by offering a cheap and rapidly produced alternative to conventional drugs. However, unlike antibacterial peptides, little is known about how these small molecules mostly exert their effects and cause death. Using live-cell imaging and deletion mutants, this study provides an analysis of the important roles that Ca2+-homeostasis and Ca2+-signalling, and possible accompanying vacuolar fusion, play during the dynamic internalization and interaction with and within the fungal cell following PAF26 treatment.