Abstract
The increasing number of life-threatening Candida infections caused by antifungal drug-resistant strains urges the development of new therapeutic strategies. The small, cysteine-rich, and cationic Neosartorya fischeri antifungal protein 2 (NFAP2) effectively inhibits the growth of Candida spp. Limiting factors of its future application, are the low-yield production by the native producer, unavailable information about potential clinical application, and the unsolved relationship between the structure and function. In the present study we adopted a Penicillium chrysogenum-based expression system for bulk production of recombinant NFAP2. Furthermore, solid-phase peptide synthesis and native chemical ligation were applied to produce synthetic NFAP2. The average yield of recombinant and synthetic NFAP2 was 40- and 16-times higher than in the native producer, respectively. Both proteins were correctly processed, folded, and proved to be heat-stable. They showed the same minimal inhibitory concentrations as the native NFAP2 against clinically relevant Candida spp. Minimal inhibitory concentrations were higher in RPMI 1640 mimicking the human inner fluid than in a low ionic strength medium. The recombinant NFAP2 interacted synergistically with fluconazole, the first-line Candida therapeutic agent and significantly decreased its effective in vitro concentrations in RPMI 1640. Functional mapping with synthetic peptide fragments of NFAP2 revealed that not the evolutionary conserved antimicrobial γ-core motif, but the mid-N-terminal part of the protein influences the antifungal activity that does not depend on the primary structure of this region. Preliminary nucleic magnetic resonance measurements signed that the produced recombinant NFAP2 is suitable for further structural investigations.
Highlights
In the last two decades, fungal infections caused by different Candida spp. have become one of the most frequent healthcare associated infections showing an increasing trend (Lockhart, 2014)
The recombinant NFAP2 (rNFAP2) was secreted into the supernatant of the P. chrysogenum nfap2 strain, from where it was purified to homogeneity
One protein band was detected by sodiumdodecyl-sulfate polyacrylamide gel electrophoresis, which corresponded to the molecular weight of native NFAP2 (nNFAP2) (Figure 1)
Summary
In the last two decades, fungal infections caused by different Candida spp. (such as candidemia and candidiasis) have become one of the most frequent healthcare associated infections showing an increasing trend (Lockhart, 2014). The Neosartorya fischeri antifungal protein 2 (NFAP2) is a novel member of small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes (Table 1). Within this protein group, NFAP2 and its putative homologs form a phylogenetically distinct clade from the well-characterized Penicillium chrysogenum antifungal protein (PAF) and Penicillium brevicompactum bubble protein (BP) homologs. The disulphide-bond stabilized and heat-resistant NFAP2 effectively inhibits the growth of Candida albicans and non-albicans Candida species in vitro possibly due to its prompt membrane disruption ability (Tóth et al, 2016). These last features render NFAP2 exceptionally suitable as potential commercial preservative, bio-pesticide, and drug against yeasts. The bulk production of NFAP2 by a “generally recognized as safe” (GRAS) microorganism or development of a chemical strategy for its low-cost synthesis is indispensable
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
