Investigation of the antimicrobial effect of neosartorya fischeri antifungal protein (NFAP) after heterologous expression in aspergillus nidulans

László Galgóczy, Laura Kovács, Zoltán Karácsony, Máté Virágh, Zsuzsanna Hamari, Csaba Vágvölgyi

Research output: Article

19 Citations (Scopus)

Abstract

Neosartorya fischeri antifungal protein (NFAP) is a β-defensin-like peptide produced by the N. fischeri NRRL 181 isolate. In this study, we investigated the manifestation of the antimicrobial effect of NFAP via heterologous expression of the nfap gene in an NFAP-sensitive fungus, Aspergillus nidulans. Heterologous expression of the nfap gene was carried out in A. nidulans CS2902 using a pAMA1-based autonomous replicative vector construct. The effect of the produced NFAP on the germination of A. nidulans conidia was investigated by scanning electron microscopy (SEM), and by DAPI and Calcofluor white (CFW) staining. 29,79Dichlorodihydrofluorescein diacetate staining and an Annexin V-FITC Apoptosis Detection kit were used to reveal the accumulation of reactive oxygen species (ROS) and the possible apoptotic, necrotic effect. The impact of monoand divalent cations on the antimicrobial activity of NFAP was also examined. Transformants expressing the nfap gene showed reduced hyphal growth compared with the untransformed strain. This effect was absent in the presence of monoand divalent cations (50 and 100 mM KCl, Mg2SO4, Na2SO4). Delayed and abnormal germination was observed in the case of transformants. Conidia developed short branching germination tubes with swollen tips. The great majority of germinating conidia were destroyed after 8 h of cultivation, although a few survived and developed into abnormal hyphae. Damage in the organization of the cell wall, the destruction of chitin filaments and the accumulation of nuclei at the broken hyphal tips were detected by SEM, DAPI and CFW staining. The accumulation of ROS and more frequent apoptotic, necrotic events were also observed in the case of the NFAPproducing A. nidulans strain.

Original languageEnglish
Pages (from-to)411-419
Number of pages9
JournalMicrobiology (United Kingdom)
Volume159
Issue number2
DOIs
Publication statusPublished - febr. 1 2013

ASJC Scopus subject areas

  • Microbiology

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