Two Functional Motifs Define the Interaction, Internalization and Toxicity of the Cell-Penetrating Antifungal Peptide PAF26 on Fungal Cells

Abstract

The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW) is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW) and PAF96 (RKKAAA), were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i) its interaction with the cell envelope; (ii) its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii) its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the cytoplasm, which coincides with cell death. Peptide containment within vacuoles preserves fungal cells from peptide toxicity.

Concatemerization increases the inhibitory activity of short, cell-penetrating, cationic and tryptophan-rich antifungal peptides

“…Some of these granules presented strong red labeling. These observations are fully consistent with previous data describing the interaction, internalization, and killing produced by PAF26 on P. digitatum, N. crassa, and S. cerevisiae (Harries et al 2013;Muñoz et al 2006Muñoz et al , 2012Muñoz et al , 2013b.…”

“…Our study shows that the kinetics of the interaction and killing of F. proliferatum by PAF102 (Figs. 6 and 7) is markedly similar to that of P. digitatum or N. crassa by PAF26 (Muñoz et al 2012(Muñoz et al , 2013b. Therefore, increase of activity is likely a consequence of the molar increase of physically linked AMP units per molecule of peptide, and not of any significant change in the mechanism of action.…”

“…The C-terminal addition of the KDEL sequence reduced 2-fold the activity in the monomeric peptides but had no significant effect in the di-or tri-PAF peptides. This result is likely due to the impact that the −1 net charge of KDEL (see Table 1), since cationic charge is critical for the initial attraction and binding of PAF peptides to the fungal surface (Muñoz et al 2013b) A positive effect was observed upon the addition of the AGPA hinge. DiPAFs and triPAFs without the AGPA tend to precipitate in some media such as the LB used in the antibacterial activity assays, as indicated by SI values above zero (Table 1), and in general do not dissolve as easily in aqueous buffers as the monomeric PAFs do.…”

“…Additional examples of fungi that have been demonstrated to be sensitive to PAF26 and PAF26 derivatives include the phytopathogens Penicillium italicum, Penicillium expansum, Botrytis cinerea, and Fusarium oxysporum (López-García et al 2002, 2003; the human pathogens Candida albicans, Aspergillus fumigatus, and several dermatophytes (López-García et al 2007;Muñoz et al 2013b); and the model fungus Neurospora crassa (Muñoz et al 2012). PAF26 is a cationic tryptophanrich peptide that belongs to the cell-penetrating class of AMPs (CP-AMPs).…”

FLO11 Gene Is Involved in the Interaction of Flor Strains of Saccharomyces cerevisiae with a Biofilm-Promoting Synthetic Hexapeptide

“…It has been shown that PAF26 is an antifungal cell-penetrating peptide that first interacts with the outer CW of fungi and that CW protein genes determine its activity toward S. cerevisiae (31,38,41). Since flor yeast strains offer a natural diversity of CW-related phenotypes associated with the abundant Flo11 protein, we decided to test the effect of PAF26 on a number of S. cerevisiae flor strains with distinct genetic backgrounds, lengths, and structure of FLO11 gene and capabilities to form biofilm (see Materials and Methods) (14).…”