Abstract
Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.
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References
Andrianopoulos A, Hynes MJ (1988) Cloning and analysis of the positively acting regulatory gene amdR from Aspergillus nidulans. Mol Cell Biol 8:3532–3541
Andrianopoulos A, Timberlake WE (1994) The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development. Mol Cell Biol 14:2503–2515
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brakhage AA (1998) Molecular regulation of beta-lactam biosynthesis in filamentous fungi. Microbiol Mol Biol Rev 62:547–585
Brakhage AA, Langfelder K (2002) Menacing mold: the molecular biology of Aspergillus fumigatus. Annu Rev Microbiol 56:433–455
Brakhage AA, Jahn B (2002) Molecular mechanisms of pathogenicity of Aspergillus fumigatus. In: Osiewacz HD (ed) Molecular biology of fungal development. Marcel Dekker, Dordrecht, pp 559–582
Brakhage AA, Sprote P, Al-Abdallah Q, Gehrke A, Plattner H, Tuncher A (2004) Regulation of penicillin biosynthesis in filamentous fungi. Adv Biochem Eng Biotechnol 88:45–90
Brakhage AA (2005) Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Curr Drug Targets 6:875–886
Brakhage AA, Liebmann B (2005) Aspergillus fumigatus conidial pigment and cAMP signal transduction: significance for virulence. Med Mycol 43 Supplement 1:S75–S82
Dutton JR, Johns S, Miller BL (1997) StuAp is a sequence-specific transcription factor that regulates development complexity in Aspergillus nidulans. EMBO J 16:5710–5721
Enjalbert B, Nantel A, Whiteway M (2003) Stress-induced gene expression in Candida albicans: absence of a general stress response. Mol Biol Cell 14:1460–1467
Haase G, Brakhage AA (2004) Melanized fungi infecting humans. In: Domer JE, Kobayashi GS (eds) Human fungal pathogens, vol. XII. Springer, Berlin Heidelberg NewYork,pp 67–88
Ibrahim-Granet O, Philippe B, Boleti H, Boisvieux-Ulrich E, Grenet D, Stern M, Latgé J-P (2003) Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages. Infect Immun 71:891–903
Jahn B, Koch A, Schmidt A, Wanner G, Gehringer H, Bhakdi S, Brakhage AA (1997) Isolation and characterisation of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence. Infect Immun 65:5110–5117
Jahn B, Boukhallouk F, Lotz J, Langfelder K, Wanner G, Brakhage AA (2000) Interaction of human phagocytes with pigmentless Aspergillus conidia. Infect Immun 68:3736–3739
Jahn B, Langfelder K, Schneider U, Schindel C, Brakhage AA (2002) PKSP-dependent reduction of phagolysosome fusion and intracellular kill of Aspergillus fumigatus conidia by human monocyte-derived macrophages. Cell Microbiol 4:793–803
Krappmann S, Sasse C, Braus GH (2006) Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background. Eukaryot Cell 5:212–215
Labbe S, Pena MM, Fernandes AR, Thiele DJ (1999) A copper-sensing transcription factor regulates iron uptake genes in Schizosaccharomyces pombe. J Biol Chem 274:36252–36260
Langfelder K, Jahn B, Gehringer H, Schmidt A, Wanner G, Brakhage AA (1998) Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med Microbiol Immunol 187:79–89
Langfelder K, Philippe B, Jahn B, Latgé J-P, Brakhage AA (2001) Differential expression of the Aspergillus fumigatus pksP gene detected in vitro and in vivo with green fluorescent protein. Infect Immun 69:6411–6418
Langfelder K, Streibel M, Jahn B, Haase G, Brakhage AA (2003) Biosynthesis of fungal melanins and their importance for human pathogenic fungi. Fungal Genet Biol 38:143–158
Latgé J-P (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12:310–350
Liebmann B, Gattung S, Jahn B, Brakhage AA (2003) cAMP signaling in Aspergillus fumigatus is involved in the regulation of the pathogenicity determinant-encoding gene pksP and the defense against killing by macrophages. Mol Gen Genom 269:420–435
Liebmann B, Mühleisen TW, Müller M, Hecht M, Weidner G, Braun A, Brock M, Brakhage AA (2004) Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis. Arch Microbiol 181:378–383
Liebmann B, Müller M, Braun A, Brakhage AA (2004) The cyclic cAMP-dependent protein kinase A network regulates development and virulence in Aspergillus fumigatus. Infect Immun 72:5193–5203
Maerker C, Rohde M, Brakhage AA, Brock M (2005) Methylcitrate synthase from Aspergillus fumigatus. Propionyl-CoA affects polyketide synthesis, growth and morphology of conidia. FEBS J 272:3615–3630
Missall T, Moran J, Corbett J, Lodge J (2005) Distinct stress responses of two functional laccases in Cryptococcus neoformans are revealed in the absence of the thiol-specific antioxidant Tsa1. Eukaryotic Cell 4:202–208
Nosanchuk J, Casadevall A (2003) The contribution of the melanin to the microbial pathogenesis. Cell Microbiol 5:203–223
Punt PJ, van den Hondel CA (1992) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Meth Enzymol 216:447–457
Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR (1996) Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med 184:377–386
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Scherer M, Fischer R (1998) Purification and characterisation of laccase II of Aspergillus nidulans. Arch Microbiol 170:78–84
Schneemann M, Schaffner A (1999) Host-defense mechanisms in Aspergillus infections. In: Brakhage AA, Jahn B, Schmidt A (eds) Contributions to Microbiol, vol 2. Karger, Basel, pp 57–68
Skromne I, Sanchez O, Aguirre J (1995) Starvation stress modulates the expression of the Aspergillus nidulans brlA regulatory gene. Microbiol 141:21–28
Smith JM, Tang CM, Van Noorden S, Holden DW (1994) Virulence of Aspergillus fumigatus double mutants lacking restrictocin and an alkaline protease in a low-dose model of invasive pulmonary aspergillosis. Infect Immun 62:5247–5254
Treger JM, Magee TR, McEntee K (1998) Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae. Biochem Biophys Res Commun 243:13–19
Trinci APJ (1971) Influence of the peripheral growth zone on the radial growth rate of fungal colonies. J Gen Microbiol 67:325–344
Tsai H-F, Chang YC, Washburn RG, Wheeler MH, Kwon-Chung KJ (1998) The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J Bacteriol 180:3031–3038
Tsai H-F, Wheeler MH, Chang YC, Kwon-Chung KJ (1999) A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus. J Bacteriol 181:6469–6477
Tsai H-F, Fujii I, Watanabe A, Wheeler MH, Chang YC, Yasuoka Y, Ebizuka Y, Kwon-Chung KJ (2001) Pentaketide-melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor. J Biol Chem 276:29292–29298
Tüncher A, Reinke H, Martic G, Caruso ML, Brakhage AA (2004) A basic-region helix-loop-helix protein-encoding gene (devR) involved in the development of Aspergillus nidulans. Mol Microbiol 52:227–241
Weidner G, d’Enfert C, Koch A, Mol PC, Brakhage AA (1998) Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′-monophosphate decarboxylase. Curr Genet 33:378–385
Zhu X, Gibbons J, Garcia- Rivera J, Casadevall A, Williamson PR (2001) Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor. Infect Immun 69:5589–5596
Zhu X, Williamson PR (2004) Role of laccase in the biology and virulence of Cryptococcus neoformans. FEMS Yeast Res 5:1–10
Acknowledgments
We thank Yvonne Speidel, Ursula Stöckel and Birgit Weber for excellent technical assistance and Sven Krappmann for providing plasmid pAN8-1. This research was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 587, Tp A8 and Priority Program 1160).
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Sugareva, V., Härtl, A., Brock, M. et al. Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus . Arch Microbiol 186, 345–355 (2006). https://doi.org/10.1007/s00203-006-0144-2
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DOI: https://doi.org/10.1007/s00203-006-0144-2