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Insight into the antifungal mechanism of Neosartorya fischeri antifungal protein.

Virágh M, Marton A, Vizler C, Tóth L, Vágvölgyi C, Marx F, Galgóczy L - Protein Cell (2015)

Bottom Line: NFAP did show neither a direct membrane disrupting-effect nor uptake by endocytosis.In contrast, NFAP does not have any influence on the cell wall integrity pathway, but an unknown cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction.Taken together, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related proteins from Aspergillus giganteus and Penicillium chrysogenum.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.

ABSTRACT
Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great potential for the development of novel antifungal strategies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the antifungal mechanism of Neosartorya fischeri antifungal protein (NFAP), a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting-effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP activates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any influence on the cell wall integrity pathway, but an unknown cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken together, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related proteins from Aspergillus giganteus and Penicillium chrysogenum.

No MeSH data available.


Related in: MedlinePlus

Physiological changes inAspergillus nidulansin the presence ofNeosartorya fischeriantifungal protein (NFAP). (A) Viability staining of Aspergillus nidulans FGSC A4 hyphae with FUN-1 dye after NFAP treatment for 30 min at 37°C. Red vacuoles A indicates metabolic activity, while green vacuoles B metabolic inactivity. (B) Propidium iodide (PI) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 16 h at 37°C. Intracellular red fluorescence indicates membrane disruption. (C) Actin distribution at A. nidulans Actin-GFP hyphal tips in response to NFAP treatment for 30 min at 30°C. C: actin patch. (D) Calcofluor white (CFW) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 30 min at 37°C. D: cap-like CFW fluorescence - site of the chitin assembly, E: lack of the cap-like CFW fluorescence, F: delocalized chitin deposition. C: untreated control, C + : positive PI staining control-hypha was treated with 70% Et-OH for 1 h at 4°C, NFAP: NFAP-treated (25 µg/mL) hyphae. Upper images, light microscopy; lower images, fluorescence microscopy of PI staining (B) and of actin distribution (C)
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Fig1: Physiological changes inAspergillus nidulansin the presence ofNeosartorya fischeriantifungal protein (NFAP). (A) Viability staining of Aspergillus nidulans FGSC A4 hyphae with FUN-1 dye after NFAP treatment for 30 min at 37°C. Red vacuoles A indicates metabolic activity, while green vacuoles B metabolic inactivity. (B) Propidium iodide (PI) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 16 h at 37°C. Intracellular red fluorescence indicates membrane disruption. (C) Actin distribution at A. nidulans Actin-GFP hyphal tips in response to NFAP treatment for 30 min at 30°C. C: actin patch. (D) Calcofluor white (CFW) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 30 min at 37°C. D: cap-like CFW fluorescence - site of the chitin assembly, E: lack of the cap-like CFW fluorescence, F: delocalized chitin deposition. C: untreated control, C + : positive PI staining control-hypha was treated with 70% Et-OH for 1 h at 4°C, NFAP: NFAP-treated (25 µg/mL) hyphae. Upper images, light microscopy; lower images, fluorescence microscopy of PI staining (B) and of actin distribution (C)

Mentions: The two-colour fluorescent FUN1 stain passively diffuses into the fungal cells and stains the cytoplasm and metabolically inactive vacuoles green, while the metabolically active vacuoles red. After short-time exposure to NFAP (30 min) reduced cellular metabolism was detected in A. nidulans FGSC A4 hyphae indicated by the presence of only green fluorescent vacuoles compared to the untreated control, which contained red fluorescent vacuoles too (Fig. 1A). This phenomenon was also observed after 60 min and 16 h of NFAP-treatment (data not shown).Figure 1


Insight into the antifungal mechanism of Neosartorya fischeri antifungal protein.

Virágh M, Marton A, Vizler C, Tóth L, Vágvölgyi C, Marx F, Galgóczy L - Protein Cell (2015)

Physiological changes inAspergillus nidulansin the presence ofNeosartorya fischeriantifungal protein (NFAP). (A) Viability staining of Aspergillus nidulans FGSC A4 hyphae with FUN-1 dye after NFAP treatment for 30 min at 37°C. Red vacuoles A indicates metabolic activity, while green vacuoles B metabolic inactivity. (B) Propidium iodide (PI) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 16 h at 37°C. Intracellular red fluorescence indicates membrane disruption. (C) Actin distribution at A. nidulans Actin-GFP hyphal tips in response to NFAP treatment for 30 min at 30°C. C: actin patch. (D) Calcofluor white (CFW) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 30 min at 37°C. D: cap-like CFW fluorescence - site of the chitin assembly, E: lack of the cap-like CFW fluorescence, F: delocalized chitin deposition. C: untreated control, C + : positive PI staining control-hypha was treated with 70% Et-OH for 1 h at 4°C, NFAP: NFAP-treated (25 µg/mL) hyphae. Upper images, light microscopy; lower images, fluorescence microscopy of PI staining (B) and of actin distribution (C)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491047&req=5

Fig1: Physiological changes inAspergillus nidulansin the presence ofNeosartorya fischeriantifungal protein (NFAP). (A) Viability staining of Aspergillus nidulans FGSC A4 hyphae with FUN-1 dye after NFAP treatment for 30 min at 37°C. Red vacuoles A indicates metabolic activity, while green vacuoles B metabolic inactivity. (B) Propidium iodide (PI) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 16 h at 37°C. Intracellular red fluorescence indicates membrane disruption. (C) Actin distribution at A. nidulans Actin-GFP hyphal tips in response to NFAP treatment for 30 min at 30°C. C: actin patch. (D) Calcofluor white (CFW) staining of A. nidulans FGSC A4 hyphae after NFAP treatment for 30 min at 37°C. D: cap-like CFW fluorescence - site of the chitin assembly, E: lack of the cap-like CFW fluorescence, F: delocalized chitin deposition. C: untreated control, C + : positive PI staining control-hypha was treated with 70% Et-OH for 1 h at 4°C, NFAP: NFAP-treated (25 µg/mL) hyphae. Upper images, light microscopy; lower images, fluorescence microscopy of PI staining (B) and of actin distribution (C)
Mentions: The two-colour fluorescent FUN1 stain passively diffuses into the fungal cells and stains the cytoplasm and metabolically inactive vacuoles green, while the metabolically active vacuoles red. After short-time exposure to NFAP (30 min) reduced cellular metabolism was detected in A. nidulans FGSC A4 hyphae indicated by the presence of only green fluorescent vacuoles compared to the untreated control, which contained red fluorescent vacuoles too (Fig. 1A). This phenomenon was also observed after 60 min and 16 h of NFAP-treatment (data not shown).Figure 1

Bottom Line: NFAP did show neither a direct membrane disrupting-effect nor uptake by endocytosis.In contrast, NFAP does not have any influence on the cell wall integrity pathway, but an unknown cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction.Taken together, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related proteins from Aspergillus giganteus and Penicillium chrysogenum.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.

ABSTRACT
Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great potential for the development of novel antifungal strategies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the antifungal mechanism of Neosartorya fischeri antifungal protein (NFAP), a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disrupting-effect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP activates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any influence on the cell wall integrity pathway, but an unknown cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken together, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related proteins from Aspergillus giganteus and Penicillium chrysogenum.

No MeSH data available.


Related in: MedlinePlus