title: Investigation of the antimicrobial effect of Neosartorya fischeri antifungal protein (NFAP) after heterologous expression in Aspergillus nidulans.
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. 2',7'-Dichlorodihydrofluorescein 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 mono- and 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 mono- and divalent cations (50 and 100 mM KCl, Mg(2)SO(4), Na(2)SO(4)). 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 NFAP-producing A. nidulans strain.
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, 6726 Közép fasor 52, Szeged, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Galgóczy
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, 6726 Közép fasor 52, Szeged, Hungary.
authors.forename: Laura
authors.initials: L
authors.lastname: Kovács
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, 6726 Közép fasor 52, Szeged, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Karácsony
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, 6726 Közép fasor 52, Szeged, Hungary.
authors.forename: Máté
authors.initials: M
authors.lastname: Virágh
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, 6726 Közép fasor 52, Szeged, Hungary.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Hamari
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, 6726 Közép fasor 52, Szeged, Hungary.
authors.forename: Csaba
authors.initials: C
authors.lastname: Vágvölgyi
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D017209:Apoptosis; D001233:Aspergillus nidulans / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D025301:Hyphae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D055326:Neosartorya / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins