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.
Highlights
Nowadays fungal infections have become a serious threat to human health and food security
In the first gene construction, the full-length AfpB coding sequence (afpB) coding sequence was cloned, while the second approach included the in silico predicted mature afpB coding sequence fused to the paf SP-pro sequence (Fig. 1a)
We described the biotechnological production and characterization of AfpB, the only antifungal proteins (AFPs) encoded in the phytopathogenic fungus P. digitatum
Summary
Nowadays fungal infections have become a serious threat to human health and food security. AFPs are small, highly stable, cationic, cysteine-rich proteins (CRPs) stabilized by up to four disulphide bridges[2] They are usually secreted in high amounts by filamentous Ascomycetes, mainly from the genera Penicillium and Aspergillus, and show potent antifungal activity against non-self fungi at micromolar concentrations. The AfpB diagram represents the genetic construction with the full-length AfpB coding sequence (in red) cloned under the control of the Ppaf and. The AfpB* diagram corresponds to the genetic construction with the in silico predicted AfpB coding sequence (afpB*) cloned under the control of the Ppaf, paf SP-pro sequence and Tpaf. (b) The PpAfpB* diagram represents the construction with the in silico predicted AfpB coding sequence (afpB*, in red) cloned under the control of the AOX1 promoter for methanol-induced expression and terminator sequences (in black) and yeast α-factor signal sequence (α-factor SS) (in green) The AfpB* diagram corresponds to the genetic construction with the in silico predicted AfpB coding sequence (afpB*) cloned under the control of the Ppaf, paf SP-pro sequence and Tpaf. (b) The PpAfpB* diagram represents the construction with the in silico predicted AfpB coding sequence (afpB*, in red) cloned under the control of the AOX1 promoter for methanol-induced expression and terminator sequences (in black) and yeast α-factor signal sequence (α-factor SS) (in green)
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