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

Abstract

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/signaling machinery. In a screen of mutants with deletions in Ca²⁺ -signaling 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 Δyvc-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.

Keywords: PAF26; antifungal peptides; antimicrobial agents; calcium signalling; mechanisms of action; resistance.