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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

Localization ofNeosartorya fischeriantifungal protein (NFAP) inAspergillus nidulans. (A) Indirect immunofluorescence staining of Aspergillus nidulans FGSC A4 hyphae with rat anti-NFAP serum and FITC-conjugated swine anti-rat IgG, after NFAP treatment for 16 h at 37°C. NFAP accumulation in A: hyphal fracture and twist; B: cell-wall bubble. C: untreated control, NFAP: NFAP-treated (25 µg/mL), NFAP + latB: NFAP- (25 µg/mL) and latrunculin B- (5 µg/mL) treated hyphae. (B) Propidium iodide (PI) staining of Aspergillus nidulans FGSC A4 hyphae after 25 µg/mL NFAP treatment for 90 min at 37°C. Red fluorescence at hyphal bubble C and around hyphal fracture D indicates membrane disruption and outflow of the hyphal/cell content. Upper images, light microscopy; lower images, fluorescence microscopy of indirect immunofluorescence staining (A) and of PI staining (B)
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Fig2: Localization ofNeosartorya fischeriantifungal protein (NFAP) inAspergillus nidulans. (A) Indirect immunofluorescence staining of Aspergillus nidulans FGSC A4 hyphae with rat anti-NFAP serum and FITC-conjugated swine anti-rat IgG, after NFAP treatment for 16 h at 37°C. NFAP accumulation in A: hyphal fracture and twist; B: cell-wall bubble. C: untreated control, NFAP: NFAP-treated (25 µg/mL), NFAP + latB: NFAP- (25 µg/mL) and latrunculin B- (5 µg/mL) treated hyphae. (B) Propidium iodide (PI) staining of Aspergillus nidulans FGSC A4 hyphae after 25 µg/mL NFAP treatment for 90 min at 37°C. Red fluorescence at hyphal bubble C and around hyphal fracture D indicates membrane disruption and outflow of the hyphal/cell content. Upper images, light microscopy; lower images, fluorescence microscopy of indirect immunofluorescence staining (A) and of PI staining (B)

Mentions: A produced polyclonal NFAP-antiserum reacted specifically with NFAP, and no signals were obtained with the control serum that was collected before the first injection (data not shown). An indirect immunofluorescence staining method was applied to study the localization of NFAP in A. nidulans FGSC A4. NFAP did not enter the fungal cell in detectable concentration levels after 30 and 60 min of exposure (data not shown), but after 16 h NFAP-specific fluorescence signals accumulated at hyphal fractures, twists and in cell-wall bubbles (Fig. 2A). To clarify whether the NFAP internalization was the consequence of an endocytic mechanism, the indirect immunofluorescence staining was repeated in the presence of 5 µg/mL latrunculin B (latB). LatB selectively inhibits the actin polymerization and therefore disturbs endocytosis. If the NFAP accumulation in the hyphae after 16 h is a consequence of an endocytotic mechanism no fluorescence signal should be observable in the cell. NFAP-specific fluorescence signals appeared in hyphae only after 16 h of incubation with NFAP and latB, but not after the exposure for 30 or 60 min (Fig. 2A). Based on these results, we conclude that NFAP is presumably not internalized by endocytosis. Instead, the accumulation of NFAP after 16 h is possibly a consequence of a passive diffusion at disrupted sites of the cell wall and plasma membrane.Figure 2


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)

Localization ofNeosartorya fischeriantifungal protein (NFAP) inAspergillus nidulans. (A) Indirect immunofluorescence staining of Aspergillus nidulans FGSC A4 hyphae with rat anti-NFAP serum and FITC-conjugated swine anti-rat IgG, after NFAP treatment for 16 h at 37°C. NFAP accumulation in A: hyphal fracture and twist; B: cell-wall bubble. C: untreated control, NFAP: NFAP-treated (25 µg/mL), NFAP + latB: NFAP- (25 µg/mL) and latrunculin B- (5 µg/mL) treated hyphae. (B) Propidium iodide (PI) staining of Aspergillus nidulans FGSC A4 hyphae after 25 µg/mL NFAP treatment for 90 min at 37°C. Red fluorescence at hyphal bubble C and around hyphal fracture D indicates membrane disruption and outflow of the hyphal/cell content. Upper images, light microscopy; lower images, fluorescence microscopy of indirect immunofluorescence staining (A) and of PI staining (B)
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Related In: Results  -  Collection

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Fig2: Localization ofNeosartorya fischeriantifungal protein (NFAP) inAspergillus nidulans. (A) Indirect immunofluorescence staining of Aspergillus nidulans FGSC A4 hyphae with rat anti-NFAP serum and FITC-conjugated swine anti-rat IgG, after NFAP treatment for 16 h at 37°C. NFAP accumulation in A: hyphal fracture and twist; B: cell-wall bubble. C: untreated control, NFAP: NFAP-treated (25 µg/mL), NFAP + latB: NFAP- (25 µg/mL) and latrunculin B- (5 µg/mL) treated hyphae. (B) Propidium iodide (PI) staining of Aspergillus nidulans FGSC A4 hyphae after 25 µg/mL NFAP treatment for 90 min at 37°C. Red fluorescence at hyphal bubble C and around hyphal fracture D indicates membrane disruption and outflow of the hyphal/cell content. Upper images, light microscopy; lower images, fluorescence microscopy of indirect immunofluorescence staining (A) and of PI staining (B)
Mentions: A produced polyclonal NFAP-antiserum reacted specifically with NFAP, and no signals were obtained with the control serum that was collected before the first injection (data not shown). An indirect immunofluorescence staining method was applied to study the localization of NFAP in A. nidulans FGSC A4. NFAP did not enter the fungal cell in detectable concentration levels after 30 and 60 min of exposure (data not shown), but after 16 h NFAP-specific fluorescence signals accumulated at hyphal fractures, twists and in cell-wall bubbles (Fig. 2A). To clarify whether the NFAP internalization was the consequence of an endocytic mechanism, the indirect immunofluorescence staining was repeated in the presence of 5 µg/mL latrunculin B (latB). LatB selectively inhibits the actin polymerization and therefore disturbs endocytosis. If the NFAP accumulation in the hyphae after 16 h is a consequence of an endocytotic mechanism no fluorescence signal should be observable in the cell. NFAP-specific fluorescence signals appeared in hyphae only after 16 h of incubation with NFAP and latB, but not after the exposure for 30 or 60 min (Fig. 2A). Based on these results, we conclude that NFAP is presumably not internalized by endocytosis. Instead, the accumulation of NFAP after 16 h is possibly a consequence of a passive diffusion at disrupted sites of the cell wall and plasma membrane.Figure 2

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