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The antifungal protein from Aspergillus giganteus causes membrane permeabilization
1Technische Universität Berlin, Institut für Biotechnologie, Fachgebiet Mikrobiologie und Genetik, 13355 Berlin, Germany.
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 micro g/ml for Fusarium oxysporum to 200 micro 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.
Insights
The antifungal protein (AFP) from Aspergillus giganteus inhibits fungal growth by permeabilizing fungal membranes. This effect is concentration-dependent and occurs rapidly, highlighting AFP as a potent antifungal agent.
Area of Science:
- Mycology
- Biochemistry
- Molecular Biology
Background:
- Filamentous fungi pose significant threats in agriculture and medicine.
- Antifungal proteins (AFPs) are a class of proteins with potential antimicrobial properties.
- Understanding the mechanism of action of AFPs is crucial for developing new antifungal strategies.
Purpose of the Study:
- To investigate the inhibitory effects of Aspergillus giganteus antifungal protein (AFP) on filamentous fungal growth.
- To elucidate the mechanism by which AFP exerts its antifungal activity.
- To determine the relationship between AFP concentration and its effects on fungal membranes.
Main Methods:
- Determination of Minimum Inhibitory Concentrations (MICs) for various fungal species.
- Assay for membrane permeabilization using SYTOX Green dye uptake.
- Fluorescence microscopy and immunofluorescence staining for protein localization.
- Investigating the effect of cations on AFP activity.
Main Results:
- AFP exhibited potent antifungal activity, with MICs varying from 0.1 µg/ml (Fusarium oxysporum) to 200 µg/ml (Aspergillus nidulans).
- AFP induced significant membrane permeabilization in sensitive fungi above the MIC, detectable within 5 minutes.
- Localization studies confirmed AFP binding to the plasma membrane of sensitive fungi, while it was found intracellularly in resistant strains.
- Antifungal activity was reduced in the presence of cations.
Conclusions:
- The growth-inhibitory effect of AFP is attributed to the permeabilization of fungal membranes.
- AFP's mechanism involves direct interaction with and disruption of the fungal cell membrane.
- Differential localization in resistant fungi suggests potential mechanisms of resistance.