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    The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis


    Gallagher, Lorna and Owens, Rebecca A. and Dolan, Stephen K. and O'Keefe, Grainne and Schrettl, Markus and Kavanagh, Kevin and Jones, Gary W. and Doyle, Sean (2012) The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis. Eukaryotic Cell, 11 (10). pp. 1226-1238. ISSN 1535-9778

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    Abstract

    The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P<0.001) and H2O2 (P<0.01), unexpectedly, exogenous gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 g/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P<0.05) the growth of the gliK26933 deletion mutant. The A. fumigatus gliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse- phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus gliK26933 mutant than in those of the wild type (P0.0024 [fold difference, 24] and P0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus gliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus gliK26933 mutant compared to the wild type (P<0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the gliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus.

    Item Type: Article
    Additional Information: The definitive version of this article is available at doi:10.1128/EC.00113-12
    Keywords: Aspergillus fumigatus Protein GliK; Oxidative Stress; Gliotoxin Biosynthesis; gliotoxin biosynthetic cluster (gli);
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 4094
    Depositing User: Dr. Kevin Kavanagh
    Date Deposited: 23 Jan 2013 16:09
    Journal or Publication Title: Eukaryotic Cell
    Publisher: American Society for Microbiology
    Refereed: Yes
    URI:

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