Abstract
Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes represent ideal bio-molecules for the development of next-generation antifungal therapeutics. They are promising candidates to counteract resistance development and may complement or even replace current small molecule-based antibiotics in the future. In this study, we show that a 14 amino acid (aa) long peptide (Pγ) spanning the highly conserved γ-core motif of the Penicillium chrysogenum antifungal protein (PAF) has antifungal activity against the opportunistic human pathogenic yeast Candida albicans. By substituting specific aa we elevated the positive net charge and the hydrophilicity of Pγ and created the peptide variants Pγvar and Pγopt with 10-fold higher antifungal activity than Pγ. Similarly, the antifungal efficacy of the PAF protein could be significantly improved by exchanging the respective aa in the γ-core of the protein by creating the protein variants PAFγvar and PAFγopt. The designed peptides and proteins were investigated in detail for their physicochemical features and mode of action, and were tested for cytotoxicity on mammalian cells. This study proves for the first time the important role of the γ-core motif in the biological function of an AMP from ascomycetes. Furthermore, we provide a detailed phylogenetic analysis that proves the presence and conservation of the γ-core motif in all AMP classes from Eurotiomycetes. We emphasize the potential of this common protein motif for the design of short antifungal peptides and as a protein motif in which targeted aa substitutions enhance antimicrobial activity.
Highlights
Fungal pathogens have a severe impact on public health
The antifungal activity of these three peptides was determined in broth microdilution assays on the opportunistic human pathogenic yeast C. albicans
Our very detailed screening and statistically well-supported phylogenetic analysis revealed that the γ-core motif is not restricted to Penicillium chrysogenum antifungal protein (PAF) and AFP homologs, but can be found in BP- and NFAP2-related proteins (Figure 8)
Summary
Fungal pathogens have a severe impact on public health. Among all opportunistic human pathogenic fungi, Candida species are the most common, whereby Candida albicans is the major representative (Brown et al, 2012). Some reports suggest that we are already on the brink of a “post-antibiotic era” as a consequence of the emerging number of resistant strains and limited number of effective antifungal agents (Kosikowska and Lesner, 2016; Kang et al, 2017). To overcome this problem, the development of new antifungal strategies is urgently needed. PAF shows potent growth inhibitory activity against numerous filamentous plant- and human-pathogenic fungi (Marx et al, 2008; Hegedüs et al, 2011), and only recently we reported on its anti-Candida effect (Huber et al, 2018)
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