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

Abstract

There are short cationic and tryptophan-rich antifungal peptides such as the hexapeptide PAF26 (RKKWFW) that have selective toxicity and cell penetration properties against fungal cells. This study demonstrates that concatemeric peptides with tandem repeats of the heptapeptide PAF54 (which is an elongated PAF26 sequence) show increased fungistatic and bacteriostatic activities while maintaining the absence of hemolytic activity of the monomer. The increase in antimicrobial activity of the double-repeated PAF sequences (diPAFs), compared to the nonrepeated PAF, was higher (4-8-fold) than that seen for the triple-repeated sequences (triPAFs) versus the diPAFs (2-fold). However, concatemerization diminished the fungicidal activity against quiescent spores of the filamentous fungus Penicillium digitatum. Peptide solubility and sensitivity to proteolytic degradation were affected by the design of the concatemers: incorporation of the AGPA sequence hinge to separate PAF54 repeats increased solubility while the C-terminal addition of the KDEL sequence decreased in vitro stability. These results led to the design of the triPAF sequence PAF102 of 30 amino acid residues, with increased antimicrobial activity and minimal inhibitory concentration (MIC) value of 1-5 μM depending on the fungus. Further characterization of the mode-of-action of PAF102 demonstrated that it colocalizes first with the fungal cell wall, it is thereafter internalized in an energy dependent manner into hyphal cells of the filamentous fungus Fusarium proliferatum, and finally kills hyphal cells intracellularly. Therefore, PAF102 showed mechanistic properties against fungi similar to the parental PAF26. These observations are of high interest in the future development of PAF-based antimicrobial molecules optimized for their production in biofactories.

Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum

“…None of the AfpB-derived peptides tested showed inhibitory effect against F. oxysporum at the maximum concentration used. This strain of F. oxysporum has shown comparatively higher tolerance to most of the peptides studied in our laboratory over the years (López-García et al, 2015). …”

“…Again, we used peptide PAF26 as control as it is considered highly resistant to protease degradation (López-García et al, 2015). The RP-HPLC chromatograms (Supplementary Figure S3) showed that all the cyclic peptides derived from AfpB were, with varying degrees, resistant to protease treatment with a high dose of enzyme (5 μg/mL) ( Figure 8 ).…”

“…The proteolytic digestion assays were performed as described (Ferre et al, 2006; López-García et al, 2015) with minor modifications. Peptides (5 μM) were dissolved in 10 mM MOPS pH 7 and digested with 5 μg/mL of recombinant proteinase K (2 U/mg; Roche, Mannheim, Germany) at 30°C.…”

Natural Antifungal Peptides/Proteins as Model for Novel Food Preservatives

“…These advances have been recently reviewed by Reinhardt and Neundorf (). The repetition of the PAF motif (rkkwfw), arranged in a repeating polymer, increased the antifungal potency compared to the monomer (López‐García et al., ). However, the solubility and the proteolytic resistance of the peptide are lowered by oligomerization (Figure ).…”

“…However, the solubility and the proteolytic resistance of the peptide are lowered by oligomerization (Figure ). This issue has been resolved through the incorporation of the AGPA sequence “hinge” between the repeats (Lopez‐Garcia et al., ). The creation of hybrids of different antifungal peptides, for example, HP(2‐9)‐magainin 2(1‐12), also increases the potency compared to the single peptides alone (Park, Lee, & Hahm, ).…”