Abstract
The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAPΔC), hydrophobic core deletion (NFAPΔh), and N-terminal amino acids exchanges (NFAPΔN) were produced in Pichia pastoris. The recombinant NFAP showed the same features in structure, folding, stability and activity as the native protein. The data acquired with mass spectrometry, structural analyses and antifungal activity assays of NFAP and its mutants proved the importance of the disulphide bonding, the hydrophobic core and the correct N-terminus for folding, stability and full antifungal function. Our findings provide further support to the comprehensive understanding of the structure-function relationship in members of this protein group.
Highlights
Recent global challenges to prevent and treat fungal infections for human welfare require the development of novel antifungal strategies against moulds[1]
Following previous observations from other cysteine-rich, β-structured antimicrobial peptides and proteins[17,18,19,20,21,22,23,24], folding, stability and antifungal activity of Neosartorya fischeri antifungal protein (NFAP) was proposed here to be dependent of the correct disulphide bond pattern, the presence of a hydrophobic core and the correct N-terminal amino acid sequence
We demonstrated that P. pastoris KM71H is able to produce folded, antifungal active NFAP, and its antifungal efficacy is comparable to the native protein[9]
Summary
Recent global challenges to prevent and treat fungal infections for human welfare require the development of novel antifungal strategies against moulds[1]. The cysteine-rich antifungal proteins from filamentous ascomycetes (AFPs) provide a feasible basis for the rational design and development of new bio-pesticides, preservatives and drugs with improved activity, efficacy, fungal-selectivity, and low-cost production[2,3,4,5] The members of this protein group show different antifungal spectra and mechanisms of action on opportunistic human, animal, plant and foodborne pathogenic filamentous fungi[6,7,8,9], but interestingly, they exhibit remarkably similar β-barrel topology constituting five highly twisted antiparallel β-strands[10,11,12,13,14]. We wanted to prove our assumption that distinct structural elements contribute to the secondary structure formation, proper folding, stability and antifungal activity of AFPs from ascomycetes like NFAP To achieve this objective, we produced recombinant NFAP mutants in P. pastoris that vary in disulphide bridge formation (NFAPΔC), the hydrophobic core (NFAPΔh) and the N-terminus (NFAPΔN) (Table 1). The folding property, structural stability, and antimicrobial activity of these NFAP mutants were investigated by thermal unfolding experiments, antifungal susceptibility tests, and functional tests and were compared with the wild-type NFAP
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