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.
Highlights
Fungal infections today are among the most difficult diseases to manage in humans (Kohler et al, 2014)
The first group of mutants screened were defective in the following Ca2+-channel proteins: CCH-1 and MID-1, which are components of the high affinity Ca2+-influx system (HACS); FIG-1, which forms the low-affinity Ca2+-influx system (LACS); and, the vacuolar Ca2+ channel, YVC-1
The ∆nca2 macroconidia internalized TMR-PAF26 at a far reduced rate compared with the other mutants and the peptide mostly became trapped within the small vesicles, resulting in virtually no TMRPAF26 being taken up by vacuoles and a correspondingly extremely small percentage (~2%) of the cells having permeabilized internal membranes after 120 min (Figure 2b)
Summary
Fungal infections today are among the most difficult diseases to manage in humans (Kohler et al, 2014). Less TMRPAF26 was taken up by ∆yvc-1 cells, most accumulated in small vesicles and few cells had internal membrane permeabilization after 120 min (5% compared with 57% in the wild type) These results are consistent with YVC-1 being required for the transport of PAF26 from vesicles to vacuoles. The ∆nca macroconidia internalized TMR-PAF26 at a far reduced rate compared with the other mutants and the peptide mostly became trapped within the small vesicles, resulting in virtually no TMRPAF26 being taken up by vacuoles and a correspondingly extremely small percentage (~2%) of the cells having permeabilized internal membranes after 120 min (Figure 2b). In the images corresponding to the second spike marked with *, the fusion of two vacuoles is clear at the top of the image
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