The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization

Abstract

We investigated the inhibitory effects of the antifungal protein (AFP) from Aspergillus giganteus on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 g/ml for Fusarium oxysporum to 200 g/ml for Aspergillus nidulans. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.The imperfect filamentous fungus Aspergillus giganteus was found to secrete two small basic proteins, namely, the antifungal protein (AFP) and ␣-sarcin (22). ␣-Sarcin is a cytotoxic protein belonging to the ribotoxins, a family of ribosome-inactivating proteins (2,6,7,13,17,24,30). AFP is a highly basic polypeptide of 51 amino acids with a high content of cysteine, tyrosine, and lysine residues. The isoelectric point was estimated to be 8.8; thus, the protein is positively charged under neutral conditions. AFP is folded into five highly twisted antiparallel strands, defining a small and compact structure with four stabilizing disulfide bridges (3,20,29).Only three small, basic antifungal proteins have been isolated from filamentous fungi to date: AFP from A. giganteus (22), PAF from Penicillium chrysogenum (18), and Anafp from Aspergillus niger (14). Recently, the naf gene was isolated from Penicillium nalgiovense and is identical to the paf gene from P. chrysogenum and encodes a protein with transient antifungal activity (8). All these antifungal proteins are secreted and inhibit the growth of numerous filamentous fungi without affecting bacteria and yeast. Most notably, they are significantly similar in regard to their structures, sizes, and basic characters. The PAF protein exhibits 42.6% sequence similarity with the AFP sequence (18).Not much is known about the modes of action of the antifungal proteins secreted by fungi. It has, however, been shown that defensins, a class of antimicrobial proteins which are quite similar to antifungal proteins in terms of their structures, sizes, and disulfide bridges, interact with anionic phospholipids of bacterial membranes (15). In addition, it has been found that some plant defensins have a receptor-mediated interaction with the fungal plasma membrane (27). However, noth...

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Bio-Control of <i>Alternaria alternata</i> during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

“…Likewise, AFP-induced membrane permeabilization was shown by the application of this technique [13]. Hence, only at concentrations that are ten-to hundred fold higher than the minimal inhibitory concentration, AFP can be detected intercellulary [12] [40].…”

“…The SYTOX-Green uptake assay was performed according to the method described by [13] [31]. One thousand spores of the fungal test strain were cultivated in 96-well microtiter plate containing 200 µl PDB medium.…”

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