title: Production of a defensin-like antifungal protein NFAP from Neosartorya fischeri in Pichia pastoris and its antifungal activity against filamentous fungal isolates from human infections.
abstract: Neosartorya fischeri NRRL 181 isolate secretes a defensin-like antifungal protein (NFAP) which has a remarkable antifungal effect against ascomycetous filamentous fungi. This protein is a promising antifungal agent of biotechnological value; however in spite of the available knowledge of the nature of its 5'-upstream transcriptional regulation elements, the bulk production of NFAP has not been resolved yet. In this study we carried out its heterologous expression in the yeast Pichia pastoris and investigated the growth inhibition effect exerted by the heterologous NFAP (hNFAP) on filamentous fungal isolates from human infections compared with what was caused by the native NFAP. P. pastoris KM71H transformant strain harboring the pPICZαA plasmid with the mature NFAP encoding gene produced the protein. The final yield of the hNFAP was sixfold compared to the NFAP produced by N. fischeri NRRL 181. Based on the signal dispersion of the amide region, it was proven that the hNFAP exists in folded state. The purified hNFAP effectively inhibited the growth of fungal isolates belonging to the Aspergillus and to the Fusarium genus, but all investigated zygomycetous strain proved to be insusceptible. There was no significant difference between the growth inhibition effect exerted by the native and the heterologous NFAP. These data indicated that P. pastoris KM71H can produce the NFAP in an antifungally active folded state. Our results provide a base for further research, e.g., investigation the connection between the protein structure and the antifungal activity using site directed mutagenesis.
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, Szeged H-6726, Hungary.
authors.forename: Máté
authors.initials: M
authors.lastname: Virágh
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, Szeged H-6726, Hungary.
authors.forename: Dóra
authors.initials: D
authors.lastname: Vörös
authors.affiliation: Department of Medical Chemistry, Faculty of Medicine, University of Szeged, Dóm tér 8, Szeged H-6720, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Kele
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, Szeged H-6726, Hungary.
authors.forename: Laura
authors.initials: L
authors.lastname: Kovács
authors.affiliation: Department of Organic Chemistry, University of Debrecen, Egyetem tér 1, Debrecen H-4010, Hungary.
authors.forename: Ádám
authors.initials: Á
authors.lastname: Fizil
authors.affiliation: Institute of Biochemistry, Hungarian Academy of Sciences, Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Lakatos
authors.affiliation: Institute of Biochemistry, Hungarian Academy of Sciences, Biological Research Centre, Temesvári krt. 62, Szeged H-6726, Hungary.
authors.forename: Gergely
authors.initials: G
authors.lastname: Maróti
authors.affiliation: Department of Organic Chemistry, University of Debrecen, Egyetem tér 1, Debrecen H-4010, Hungary.
authors.forename: Gyula
authors.initials: G
authors.lastname: Batta
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, Szeged H-6726, Hungary.
authors.forename: Csaba
authors.initials: C
authors.lastname: Vágvölgyi
authors.affiliation: Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, Szeged H-6726, Hungary. Electronic address: galgoczi@gmail.com.
authors.forename: László
authors.initials: L
authors.lastname: Galgóczy
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D023082:Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D003881:Dermatomycoses / Q000382:microbiology; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D005658:Fungi / Q000187:drug effects* / Q000472:pathogenicity; D006801:Humans; D055326:Neosartorya / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics
chemical_list: D000935:Antifungal Agents; D023082:Defensins; D005656:Fungal Proteins
keywords: Antimicrobial susceptibility; Ascomycetes; Clinical fungal isolates; Neosartorya fischeri antifungal protein; Pichia pastoris; Zygomycetes