Transcriptome analysis of Aspergillus fumigatus exposed to voriconazole
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.
KeywordsAspergillus fumigatus Voriconazole Microarrays Transcriptome
We would like to thank the Fundação de Amparo a Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico, both from Brazil, for financial support for our research, and SPP1160 of the Deutsche Forschungsgemeinschaft for A.A.B. Microarray analysis work was supported by NIAID grant to W.C.N. We also thank the two anonymous reviewers and the editor for the suggestions.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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