13Fungal diseases are responsible for the deaths of over 1.5 million people worldwide 14 annually. Antifungal peptides represent a useful source of antifungals with novel 15 mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here 16 we investigate the mode-of-action of the small, rationally designed synthetic antifungal 17 peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell 18 killing activity of PAF26 is dependent on extracellular Ca 2+ and the presence of fully 19 functioning fungal Ca 2+ homeostatic/signalling machinery. In a screen of mutants with 20 deletions in Ca 2+ -signalling machinery, we identified three mutants more tolerant to PAF26. 21 The Ca 2+ ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the 22 cell envelope. The vacuolar Ca 2+ channel YVC-1 was shown to be essential for its 23 accumulation and concentration within the vacuolar system. The Ca 2+ channel CCH-1 was 24 found to be required to prevent the translocation of PAF26 across the plasma membrane. In 25 the wild type, Ca 2+ removal from the medium resulted in the peptide remaining trapped in 26 small vesicles as in the Δ yvc-1 mutant. It is therefore apparent that cell killing by PAF26 is 27 complex and unusually dependent on extracellular Ca 2+ and components of the Ca 2+ -28 regulatory machinery. 29
AUTHOR SUMMARY
30Life threatening diseases can be caused when fungi invade human tissues. These invasions 31 often occur when a person's immune defences are down, often due to treatments for cancer 32 or transplantation. These infections are commonly buried deep within the body and as such 33 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 35 infections effectively untreatable -a death sentence. Antifungal peptides such as PAF26 36 provide a possible solution by offering a cheap and rapidly produced alternative to 37 conventional drugs. However, unlike antibacterial peptides, little is known about how these 38 small molecules mostly exert their effects and cause death. Using live-cell imaging and 39 deletion mutants, this study provides an analysis of the important roles that Ca 2+ -40 homeostasis and Ca 2+ -signalling, and possible accompanying vacuolar fusion, play during 41 the dynamic internalization and interaction with and within the fungal cell following PAF26 42 treatment. 43 44 48 Denning et al. 2012, Bongomin, Gago et al. 2017. Increasing resistance to the limited 49 arsenal of antifungal drugs is a serious concern, especially for Candida and Aspergillus 50 infections, for which the therapeutic options are limited. Overall, there is an urgent need to 51 develop new antifungal strategies to tackle fungal infections (Denning and Bromley 2015, 52 Nicola, Albuquerque et al. 2019). 53 Antifungal peptides (AFPs) and peptide-related molecules are being intensively 54 studied as alternatives for the therapeutic...