title: Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence.
abstract: Aspergillus fumigatus is a saprophytic mold that can cause life-threatening infections in immunocompromised patients. In the lung, inhaled conidia are confronted with immune effector cells that attack the fungus by various mechanisms such as phagocytosis, production of antimicrobial proteins or generation of reactive oxygen intermediates. Macrophages and neutrophils can also form nitric oxide (NO) and other reactive nitrogen intermediates (RNI) that potentially also contribute to killing of the fungus. However, fungi can produce several enzymes involved in RNI detoxification. Based on genome analysis of A. fumigatus, we identified two genes encoding flavohemoglobins, FhpA, and FhpB, which have been shown to convert NO to nitrate in other fungi, and a gene encoding S-nitrosoglutathione reductase GnoA reducing S-nitrosoglutathione to ammonium and glutathione disulphide. To elucidate the role of these enzymes in detoxification of RNI, single and double deletion mutants of FhpA, FhpB, and GnoA encoding genes were generated. The analysis of mutant strains using the NO donor DETA-NO indicated that FhpA and GnoA play the major role in defense against RNI. By generating fusions with the green fluorescence protein, we showed that both FhpA-eGFP and GnoA-eGFP were located in the cytoplasm of all A. fumigatus morphotypes, from conidia to hyphae, whereas FhpB-eGFP was localized in mitochondria. Because fhpA and gnoA mRNA was also detected in the lungs of infected mice, we investigated the role of these genes in fungal pathogenicity by using a murine infection model for invasive pulmonary aspergillosis. Remarkably, all mutant strains tested displayed wild-type pathogenicity, indicating that the ability to detoxify host-derived RNI is not essential for virulence of A. fumigatus in the applied mouse infection model. Consistently, no significant differences in killing of ΔfhpA, ΔfhpB, or ΔgnoA conidia by cells of the macrophage cell line MH-S were observed when compared to the wild type.
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Lapp
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Vödisch
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Kroll
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Strassburger
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany ; Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena Germany ; Integrated Research Treatment-Center - Center for Sepsis Control and Care, University Hospital Jena, Jena Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Kniemeyer
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany ; Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Heinekamp
authors.affiliation: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute, Jena Germany ; Department of Microbiology and Molecular Biology, Institute of Microbiology, Friedrich Schiller University Jena, Jena Germany.
authors.forename: Axel A
authors.initials: AA
authors.lastname: Brakhage
keywords: Aspergillus fumigatus; nitric oxide; reactive nitrogen intermediates; virulence