Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi

The tyrocidines and analogues are cyclic decapeptides produced by Brevibacillus parabrevis with a conserved sequence of cyclo(D-Phe1-Pro2-X3-x4-Asn5-Gln6-X7-Val8-X9-Leu10) with Trp3,4/Phe3,4 in the aromatic dipeptide unit, Lys9/Orn9 as their cationic residue and Tyr (tyrocidines), Trp (tryptocidines) or Phe (phenicidines) in position 7. Previous studies indicated they have a broad antifungal spectrum with the peptides containing a Tyr residue in position 7 being more active than those with a Phe or Trp residue in this position. Detailed analysis of antifungal inhibition parameters revealed that Phe3-D-Phe4 in the aromatic dipeptide unit lead to more consistent activity against the three filamentous fungi in this study. These peptides exhibited high membrane activity and fast leakage kinetics against model membranes emulating fungal membranes, with selectivity towards ergosterol containing membranes. More fluid membranes and doping of liposomes with the sphingolipid, glucosylceramide, led to a decreased permeabilising activity. Peptide-induced uptake of membrane impermeable dyes was observed in hyphae of both Fusarium solani and Botrytis cinerea, with uptake more pronounced at the hyphal growth tips that are known to contain ergosterol-sphigolipid rich lipid rafts. Tyrocidine interaction with these rafts may lead to the previously observed fungal hyperbranching. However, the leakage of model membranes and Bot. cinerea did not correlate directly with the antifungal inhibition parameters, indicating another target or mode of action. Proteinase K treatment of target fungi had a minimal influence or even improved the tyrocidine activity, ruling out a mannoprotein target in the fungal cell wall. β-glucanase treatment of Bot. cinerea did not significantly affect the tyrocidine activity, but there was a significant loss in activity towards the β-glucanase treated F. solani. This study showed the tyrocidine antifungal membrane activity is selective towards ergosterol and possibly lipid rafts, but also point to additional targets such as the cell wall β-glucans that could modulate their activity.