Efficient production and characterization of the novel and highly active antifungal protein AfpB from Penicillium digitatum

Abstract

Filamentous fungi encode distinct antifungal proteins (AFPs) that offer great potential to develop new antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum, the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene (afpB), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P. digitatum strains. Biotechnological production and secretion of AfpB were achieved in P. digitatum with the use of a P. chrysogenum-based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed antifungal activity higher than the P. chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P. digitatum, which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi.

Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus

“…Moreover, ECFs enriched in AgAFP showed antifungal activity against P. digitatum (Figure c). This activity seems to be at least one order of magnitude lower than that of the ECFs containing AfpB (Figure c), in agreement with the high activity reported for AfpB (Garrigues et al ., ). These results, obtained with a different AFP, indicate that our TMV‐based system can be considered a general platform for producing antifungal proteins in biofactory plants.…”

“…This is a remarkable result because these small cysteine‐rich proteins have been shown to be difficult to produce in a soluble, correctly folded conformation in bacterial systems (Kiedzierska et al ., ; Rosano and Ceccarelli, ). They only had been successfully produced as active proteins in Pichia pastori at relatively low yields (Garrigues et al ., ; López‐García et al ., ; Virágh et al ., ), and at better yields in filamentous fungi (Garrigues et al ., ; Huber et al ., ; Sonderegger et al ., ; Tóth et al ., ). Although plant and fungal systems are similar in terms of yield and activity, the transient production of AFPs in plants represents an attractive alternative as sustainable bioproduction factories fueled by sunlight and easily scalable.…”

“…AfpB is an AFP whose gene was identified in the genome of the postharvest phytopathogenic fungus P. digitatum but that, unlike other AFPs, is not naturally produced by the fungus . In a recent study, P. digitatum was genetically modified to produce AfpB and was found to be a highly active antifungal protein, with inhibitory concentrations up to one order of magnitude lower than other AFPs (Garrigues et al, 2017). Therefore, there is a great interest in obtaining large amounts of AfpB, so we assessed this possibility using our new TMV vector system.…”

“…Antifungal proteins (AFPs) produced by filamentous fungi are a specific class of antimicrobial peptides (AMPs). Antifungal proteins are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture (Garrigues et al, 2017;Huber et al, 2018;L opez-Garc ıa et al, 2012;Meyer, 2008). AFPs are small proteins, usually cationic, that are rich in cysteine residues, and are folded in compact structures supported by disulphide bridges, which make them highly stable and resistant to heat, proteases and extreme pH (Batta et al, 2009;Campos-Olivas et al, 1995;Garrigues et al, 2017;Heged€ us and Marx, 2013).…”

“…Antifungal proteins are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture (Garrigues et al, 2017;Huber et al, 2018;L opez-Garc ıa et al, 2012;Meyer, 2008). AFPs are small proteins, usually cationic, that are rich in cysteine residues, and are folded in compact structures supported by disulphide bridges, which make them highly stable and resistant to heat, proteases and extreme pH (Batta et al, 2009;Campos-Olivas et al, 1995;Garrigues et al, 2017;Heged€ us and Marx, 2013). They exhibit potent specific antifungal activity at very low concentrations against important human and plant fungal pathogens (Garrigues et al, 2017;Huber et al, 2018;Marx et al, 2008;T oth et al, 2016;Vila et al, 2001;Vir agh et al, 2014), and do not have toxic effects on plant or mammalian cells (Moreno et al, 2006;Szappanos et al, 2006).…”