Abstract
Antifungal proteins of fungal origin (AFPs) are small, secreted, cationic, and cysteine-rich proteins. Filamentous fungi encode a wide repertoire of AFPs belonging to different phylogenetic classes, which offer a great potential to develop new antifungals for the control of pathogenic fungi. The fungus Penicillium expansum is one of the few reported to encode three AFPs each belonging to a different phylogenetic class (A, B, and C). In this work, the production of the putative AFPs from P. expansum was evaluated, but only the representative of class A, PeAfpA, was identified in culture supernatants of the native fungus. The biotechnological production of PeAfpB and PeAfpC was achieved in Penicillium chrysogenum with the P. chrysogenum-based expression cassette, which had been proved to work efficiently for the production of other related AFPs in filamentous fungi. Western blot analyses confirmed that P. expansum only produces PeAfpA naturally, whereas PeAfpB and PeAfpC could not be detected. From the three AFPs from P. expansum, PeAfpA showed the highest antifungal activity against all fungi tested, including plant and human pathogens. P. expansum was also sensitive to its self-AFPs PeAfpA and PeAfpB. PeAfpB showed moderate antifungal activity against filamentous fungi, whereas no activity could be attributed to PeAfpC at the conditions tested. Importantly, none of the PeAFPs showed hemolytic activity. Finally, PeAfpA was demonstrated to efficiently protect against fungal infections caused by Botrytis cinerea in tomato leaves and Penicillium digitatum in oranges. The strong antifungal potency of PeAfpA, together with the lack of cytotoxicity, and significant in vivo protection against phytopathogenic fungi that cause postharvest decay and plant diseases, make PeAfpA a promising alternative compound for application in agriculture, but also in medicine or food preservation.
Highlights
Fungal infections are an emerging worldwide threat to animal, human, and wildlife health (Fisher et al, 2012; Meyer et al, 2016)
In order to detect and isolate any of the three putative P. expansum Antifungal proteins of fungal origin (AFPs), called PeAfpA, PeAfpB, and PeAfpC, from culture supernatants, the fungus was grown in either Potato Dextrose Broth (PDB) or P. chrysogenum minimal medium (PcMM) growth media, and time-course supernatants were analyzed by SDS-PAGE (Figure 1A)
The largest amount of proteins was detected in PcMM supernatants, from which a protein band of apparent molecular mass of approximately 6 kDa was observed from day 5 till day 10 of growth
Summary
Fungal infections are an emerging worldwide threat to animal, human, and wildlife health (Fisher et al, 2012; Meyer et al, 2016). Control of pathogenic fungi represents a serious challenge due to the increasing number of immunocompromised patients and the emergence of antifungal resistant strains. Newly developed antimycotics should combine major aspects such as sustainability, high efficacy, limited toxicity, and low costs of production (Marx et al, 2008; Meyer, 2008). Antifungal proteins (AFPs) secreted by filamentous fungi meet the desired characteristics to fight fungal contaminations and infections. AFPs are small, cationic, cysteine-rich proteins highly stable to pH, high temperatures, and proteolysis, and exhibit broad antifungal spectra and different mechanisms of action against opportunistic human, animal, plant, and foodborne pathogenic filamentous fungi (Marx et al, 2008; Hegedüs and Marx, 2013; Delgado et al, 2016). AFPs are coded with a signal peptide (SP) at the N-termini that includes a pre-sequence involved in AFP secretion to the extracellular space, and a prosequence, whose function is still controversial it is assumed that might be involved in maintaining AFPs in an inactive form (Marx et al, 1995)
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