Current Genetics

, Volume 50, Issue 1, pp 32–44 | Cite as

Transcriptome analysis of Aspergillus fumigatus exposed to voriconazole

  • Márcia Eliana da Silva Ferreira
  • Iran Malavazi
  • Marcela Savoldi
  • Axel A. Brakhage
  • Maria Helena S. Goldman
  • H. Stanley Kim
  • William C. Nierman
  • Gustavo H. Goldman
Research Article

Abstract

For a comprehensive evaluation of genes that have their expression modulated during exposure of the mycelia to voriconazole, we performed a large-scale analysis of gene expression in Aspergillus fumigatus using a microarray hybridization approach. By comparing the expression of genes between the reference time and after addition of voriconazole (30, 60, 120, and 240 min), we identified 2,271 genes differentially expressed in the wild-type strain. To validate the expression of some of these genes during exposure to voriconazole, we analyzed 13 genes showing higher expression in the presence of voriconazole by real-time RT-PCR. Although the magnitudes of induction differed between the two experimental systems, in about 85% of the cases they were in good agreement with the microarray data. To our knowledge this is the first study of microarray hybridization analysis for a filamentous fungus exposed to an antifungal agent. In our study, we have observed: (i) a decreased mRNA expression of various ergosterol biosynthesis genes; (ii) increased mRNA levels of genes involved in a variety of cell functions, such as transporters, transcription factors, proteins involved in cell metabolism, and hypothetical proteins; and (iii) the involvement of the cyclic AMP-protein kinase signaling pathway in the increased mRNA expression of several of these genes.

Keywords

Aspergillus fumigatus Voriconazole Microarrays Transcriptome 

Supplementary material

References

  1. Agarwal AK, Rogers PD, Baerson SR, Jacob MR, Barker KS, Cleary JD, Walker LA, Nagle DG, Clark AM (2003) Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae. J Biol Chem 12:34998–35015CrossRefGoogle Scholar
  2. Bammert GF, Fostel JM (2000) Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol. Antimicrob Agents Chemother 44:1255–1265PubMedCrossRefGoogle Scholar
  3. Barker KS, Crisp S, Wiederhold N, Lewis RE, Bareither B, Eckstein J, Barbuch R, Bard M, Rogers PD (2004) Genome-wide expression profiling reveals genes associated with amphotericin B and fluconazole resistance in experimentally induced antifungal resistant isolates of Candida albicans. J Antimicrob Chemother 54:376–385PubMedCrossRefGoogle Scholar
  4. Burns C, Geraghty R, Neville C, Murphy A, Kavanagh K, Doyle S (2005) Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus. Fungal Genet Biol 42:319–327PubMedCrossRefGoogle Scholar
  5. Dannaoui E, Borel E, Monier MF, Piens MA, Picot S, Persat F (2001) Acquired itraconazole resistance in Aspergillus fumigatus. J Antimicrob Chemother 47:333–340PubMedCrossRefGoogle Scholar
  6. De Backer MD, Ilyina T, Ma XJ, Vandoninck S, Luyten WH, Vanden Bossche H (2001) Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray. Antimicrob Agents Chemother 45:1660–1670PubMedCrossRefGoogle Scholar
  7. Denning DW (1996) Diagnosis and management of invasive aspergillosis. Curr Clin Top Infect Dis 16:277–299PubMedGoogle Scholar
  8. Denning DW, Venkateswarlu K, Oakley KL, Anderson MJ, Manning NJ, Stevens DA, Warnock DW, Kelly SL (1997) Itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 41:1364–1368PubMedGoogle Scholar
  9. Diaz-Guerra TM, Mellado E, Cuenca-Estrella M, Rodríguez-Tudela JL (2003) A point mutation in the 14α-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 47:1120–1124PubMedCrossRefGoogle Scholar
  10. Espinel-Ingroff A, Fothergill A, Ghannoum M, Manavathu E, Ostrosky-Zeichner L, Pfaller M, Rinaldi M, Schell W, Walsh T (2005) Quality control and reference guidelines for CLSI broth microdilution susceptibility method (M 38-A document) for amphotericin B, itraconazole, posaconazole, and voriconazole. J Clin Microbiol 43:5243–5246PubMedCrossRefGoogle Scholar
  11. Ferreira ME, Colombo AL, Paulsen I, Ren Q, Wortman J, Huang J, Goldman MH, Goldman GH (2005) The ergosterol biosynthesis pathway, transporter genes, and azole resistance in Aspergillus fumigatus. Med Mycol 43:S313–S319PubMedCrossRefGoogle Scholar
  12. Hanson PI, Whiteheart SW (2005) AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol 6:519–529PubMedCrossRefGoogle Scholar
  13. Herbrecht R, Denning D, Patterson TF, Bennett JE, Greene RE, Oestmann JW, Kern WV, Marr KA, Ribaud P, Lortholary O, Silvestre R, Rubin RH, Wingard JR, Stark P, Durand C, Caillot D, Thiel E, Chandrasekar PH, Hodges MR, Schlamm HT, Troke PF, de Pauw B (2002) Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 347:408–415PubMedCrossRefGoogle Scholar
  14. Jain P, Akula I, Edlind T (2003) Cyclic AMP signaling pathway modulates susceptibility of candida species and Saccharomyces cerevisiae to antifungal azoles and other sterol biosynthesis inhibitors. Antimicrob Agents Chemother 47:3195–3201PubMedCrossRefGoogle Scholar
  15. Kafer E (1977) Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet 19:33–131PubMedCrossRefGoogle Scholar
  16. Karababa M, Coste AT, Rognon B, Bille J, Sanglard D (2004) Comparison of gene expression profiles of Candida albicans azole-resistant clinical isolates and laboratory strains exposed to drugs inducing multidrug transporters. Antimicrob Agents Chemother 48:3064–3079PubMedCrossRefGoogle Scholar
  17. Kelly SL, Lamb DC, Kelly DE, Loeffler J, Einsele H (1996) Resistance to fluconazole and amphotericin in Candida albicans from AIDS patients. Lancet 348:1523–1524PubMedCrossRefGoogle Scholar
  18. Kelly SL, Lamb DC, Kelly DE, Manning NJ, Loeffler J, Hebart H, Schumacher U, Einsele H (1997) Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta 5,6-desaturation. FEBS Lett 400:80–82PubMedCrossRefGoogle Scholar
  19. Kontoyiannis DP, Rupp S (2000) Cyclic AMP and fluconazole resistance in Saccharomyces cerevisiae. Antimicrob Agents Chemother 44:1743–1744PubMedCrossRefGoogle Scholar
  20. Krappmann S, Bignell EM, Reichard U, Rogers T, Haynes K, Braus GH (2004) The Aspergillus fumigatus transcriptional activator CpcA contributes significantly to the virulence of this fungal pathogen. Mol Microbiol 52:785–799PubMedCrossRefGoogle Scholar
  21. Langfelder K, Gattung S, Brakhage AA (2002) A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene. Curr Genet 41:268–274PubMedCrossRefGoogle Scholar
  22. Liebmann B, Gattung S, Jahn B, Brakhage AA (2003) cAMP signaling in Aspergillus fumigatus is involved in the regulation of the virulence gene pksP and in defense against killing by macrophages. Mol Genet Genomics 269:420–435PubMedCrossRefGoogle Scholar
  23. Liebmann B, Muller M, Braun A, Brakhage AA (2004) The cyclic AMP-dependent protein kinase a network regulates development and virulence in Aspergillus fumigatus. Infect Immun 72:5193–5203PubMedCrossRefGoogle Scholar
  24. Liu TT, Lee RE, Barker KS, Lee RE, Wei L, Homayouni R, Rogers PD (2005) Genome-wide expression profiling of the response to azole, polyene, echinocandin, and pyrimidine antifungal agents in Candida albicans. Antimicrob Agents Chemother 49:2226–2236PubMedCrossRefGoogle Scholar
  25. Lupetti A, Danesi R, Campa M, del Tacca M, Kelly S (2002) Molecular basis of resístance to azole antifungals. Trends Mol Med 8:76–81PubMedCrossRefGoogle Scholar
  26. Manavathu EK, Vazquez JA, Chandrasekar PH (1999) Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent. Int J Antimicrob Agents 12:213–219PubMedCrossRefGoogle Scholar
  27. Mann PA, Parmegiani RM, Wei S-Q, Mendrick CA, Li X, Loenberg D, DiDomenico B, Hare RS, Walker SS, McNicholas PM (2003) Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P-450 14α-demethylase. Antimicrob Agents Chemother 47:577–581PubMedCrossRefGoogle Scholar
  28. Marichal P, Koymas L, Willlemsens S, Bellens D, Verhasselt P, Luyten W, Borgers M, Ramaekers FCS, Odds FC, VandenBossche H (1999) Contribution of mutations in the cytochrome P-450 14-α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans. Microbiology 145:2701–2713PubMedGoogle Scholar
  29. Mellado E, Diaz-Guerra TM, Cuenca-Estrella M, Rodriguez-Tudela JL (2001) Identification of two different 14-alpha sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species. J Clin Microbiol 39:2431–2438 (Erratum in: J Clin Microbiol 2001; 39:4225)Google Scholar
  30. Nascimento AM, Goldman GH, Park S, Marras SA, Delmas G, Oza U, Lolans K, Dudley MN, Mann PA, Perlin DS (2003) Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole. Antimicrob Agents Chemother 47:1519–1526CrossRefGoogle Scholar
  31. National Committee for Clinical Laboratory Standards (2002) Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard M38-A. National Committee for Clinical Laboratory Standards, WayneGoogle Scholar
  32. Nolte FS, Parkinson T, Falconer DJ, Dix S, Williams J, Gilmore C, Geller R, Wingard JR (1997) Isolation and characterization of fluconazole- and amphotericin B-resistant Candida albicans from blood of two patients with leukaemia. Antimicrob Agents Chemother 44:196–199Google Scholar
  33. Osherov N, Kontoyannis DP, Romans A, May GS (2001) Resistance to itraconazole in Aspergillus nidulans and Aspergillus fumigatus is conferred by extra copies of the A. nidulans P-450 14α-demethylase gene, pdmA. J Antimicrob Chemother 48:75–81PubMedCrossRefGoogle Scholar
  34. Ramesh MA, Laidlaw RD, Durrenberger F, Orth AB, Kronstad JW (2001) The cAMP signal transduction pathway mediates resistance to dicarboximide and aromatic hydrocarbon fungicides in Ustilago maydis. Fungal Genet Biol 32:183–193PubMedCrossRefGoogle Scholar
  35. Rogers PD, Barker KS (2003) Genome-wide expression profile analysis reveals coordinately regulated genes associated with stepwise acquisition of azole resistance in Candida albicans clinical isolates. Antimicrob Agents Chemother 47:1220–1227PubMedCrossRefGoogle Scholar
  36. Sanglard D, Odds FC (2002) Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2:73–85PubMedCrossRefGoogle Scholar
  37. Sanglard D, Ischer F, Parkinson T, Falconer D, Bille J (2003a) Candida albicans: mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents Chemother 47:2404–2412CrossRefGoogle Scholar
  38. Sanglard D, Ischer F, Marchetti O, Entenza J, Bille J (2003b) Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Mol Microbiol 48:959–976CrossRefGoogle Scholar
  39. Semighini CP, Marins M, Goldman MHS, Goldman GH (2002) Quantitative analysis of the relative transcript levels of ABC transporter Atr genes in Aspergillus nidulans by real-time reverse transcription-PCR assay. Appl Environ Microbiol 68:1351–1357PubMedCrossRefGoogle Scholar
  40. Slaven JW, Anderson MJ, Sanglard D, Dixon GK, Bille J, Roberts IS, Denning DW (2002) Increased expression of a novel Aspergillus fumigatus ABC transporter gene, AtrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genet Biol 36:199–206PubMedCrossRefGoogle Scholar
  41. Tobin MB, Peery RB, Skatrud PL (1997) Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus. Gene 200:11–23PubMedCrossRefGoogle Scholar
  42. Tsitsigiannis DI, Kowieski TM, Zarnowski R, Keller NP (2005a) Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans. Microbiology 151:1809–1821CrossRefGoogle Scholar
  43. Tsitsigiannis DI, Bok JW, Andes D, Nielsen KF, Frisvad JC, Keller NP (2005b) Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence. Infect Immun 73:4548–4559CrossRefGoogle Scholar
  44. White TC, Marr KA, Bowden RA (1998) Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11:382–402PubMedGoogle Scholar
  45. Zhang L, Zhang Y, Zhou Y, An S, Zhou Y, Cheng J (2002) Response of gene expression in Saccharomyces cerevisiae to amphotericin B and nystatin measured by microarrays. J Antimicrob Chemother 49:905–915PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Márcia Eliana da Silva Ferreira
    • 1
  • Iran Malavazi
    • 1
  • Marcela Savoldi
    • 1
  • Axel A. Brakhage
    • 2
  • Maria Helena S. Goldman
    • 3
  • H. Stanley Kim
    • 4
    • 5
  • William C. Nierman
    • 4
    • 5
  • Gustavo H. Goldman
    • 1
  1. 1.Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão PretoUniversidade de São PauloRibeirão PretoBrazil
  2. 2.Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research, Infection Biology-HansKnoell Institute (HKI)University of JenaJenaGermany
  3. 3.Faculdade de Filosofia, Ciências e Letras de Ribeirão PretoUniversidade de São PauloRibeirão PretoBrazil
  4. 4.The Institute for Genomic ResearchRockvilleUSA
  5. 5.Department of Biochemistry and Molecular BiologyThe George Washington University School of MedicineWashingtonUSA

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