Cysteine-Rich Antifungal Proteins from Filamentous Fungi are Promising Bioactive Natural Compounds in Anti- Candida Therapy

Abstract

The emerging number of life-threatening invasive fungal infections caused by drug-resistant Candida strains urges the need for the development and application of fundamentally new and safe antifungal strategies in the clinical treatment. Recent studies demonstrated that the extracellular cysteine-rich and cationic antifungal proteins (crAFPs) originating from filamentous fungi, and de novo designed synthetic peptide derivatives of these crAFPs provide a feasible basis for this approach. This mini-review focuses on the global challenges of the anti-Canidia therapy and on the crAFPs as potential drug candidates to overcome existing problems. The advantages and limitations in the use of crAFPs and peptide derivatives compared to those of conventional antifungal drugs will also be critically discussed.

Keywords: Candida spp.; antifungal protein; drug-resistance; licensed antifungal drug; synthetic peptide.

Figure 1

Primary structure of anti‐yeast crAFPs isolated from Eurotiomycetes. ClustalW multiple alignment of the prepro‐sequences and the sequences of mature proteins were generated with the BioEdit program36 and visualized with Jalview version 2.10.3b1.37 The cleavage site of the predicted signal sequence (pre‐sequence indicated by grey line) was predicted by SignalP1 4.1 server.38 Connective orange lines between Cys residues (C in black frame) indicate disulfide bridge formations. The ClustalX default color scheme was applied in the alignment (http://www.jalview.org/help/html/colourSchemes/clustal.html) to show similarity.37 Brackets in the sequence of PAFB indicate that this protein is expressed in 58 amino acid full‐length form into the supernatant, however the leucine (L) and the serine (S) are cleaved from the N‐terminus with time, generating the 56 amino acid sfPAF form which served for the tertiary structure determination.28

Figure 2

Secondary structure and relative solvent accessibility (RSA) of anti‐yeast crAFPs isolated from Eurotiomycetes. Linear representation of secondary structures and RSA were generated from the respective annotated (PAFB, PAF, BP) or predicted (AnAFP and FPAP) .pdb file of tertiary structure (Protein Data Bank IDs: PAFB‐2nc2, PAF‐2mhv, BP‐1uoy) with POLYVIEW‐2D server39 and revised based on the tertiary structure visualized with UCSF Chimera software.40 Blue lines and green arrows indicate the loop and β‐strand regions, respectively. Connective orange lines between Cys residues indicate disulfide bridge formations. Below the secondary structure, the RSA is indicated, in which the black and white squares represent completely buried (0‐9 RSA) and fully exposed (90‐100 RSA) amino acids, respectively. Putative tertiary structure of AnAFP and FPAP was predicted in silico by I‐Tasser,^[41] and refined by ModRefiner.42 Tertiary structure of PAF (Protein Data Bank ID: 2mhv) served as a template to model the structure of AnAFP and FPAP. Question marks indicate that the secondary structure of NFAP2 is under investigation.

Figure 3

Tertiary structure, hydrophobicity (Kyte‐Doolittle scale of amino acids with colors ranging from blue for the most hydrophilic to white at 0.0 to orange for the most hydrophobic) and electrostatic surface (Coulombic surface coloring of amino acids with colors ranging from dark blue for positive charge to white at 0 to red for negative charge) (from up to down) of anti‐yeast crAFPs isolated from Eurotiomycetes. The first loop region and the γ‐core motif is indicated by black and grey, respectively. Cys residues and disulfide bridges are marked in yellow and yellow lines, respectively. All structures were generated with UCSF Chimera visualization software using the respective annotated (Protein Data Bank IDs: PAFB‐2nc2, PAF‐2mhv, BP‐1uoy) or predicted (AnAFP and FPAP) .pdf file of tertiary structure, respectively.40 Putative tertiary structure of AnAFP and FPAP was predicted in silico by I‐Tasser,41 and refined by ModRefiner.42 Based on the Ramachandran plot analysis,43 96.4 % and 100 % of the residues are in the favored and allowed position in AnAFP and FPAP, respectively. Tertiary structure of PAF (Protein Data Bank ID: 2mhv) served as a template to model the structure of AnAFP and FPAP. Question marks indicate that the tertiary structure, hydrophobicity and electrostatic surface of NFAP2 are under investigation.