title: Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine: genetic approach to studying gene dose-dependent resistance to antifungals in A. fumigatus.
abstract: With the increasing use of antifungals such as amphotericin B, itraconazole, voriconazole, caspofungin, and terbinafine (TRB) in patients at high risk for invasive aspergillosis, resistance of Aspergillus fumigatus to these agents will ultimately emerge. Due to the limited availability of molecular genetics for A. fumigatus, few studies have addressed its mechanisms of resistance to antifungals. We transformed A. fumigatus protoplasts with a pyrG-based A. fumigatus genomic DNA library (constructed in the multicopy nonintegrating vector pRG3-AMA1-NotI, which also has the pyr-4 gene for selection). We obtained one pyrG(+) transformant that grew in medium containing a fungicidal concentration (0.625 microg/ml) of TRB. To determine whether TRB resistance in that transformant was plasmid dependent, we evicted the plasmid and found concomitant loss of uracil prototrophy and TRB resistance. DNA sequence analysis identified the gene responsible for TRB resistance as the A. fumigatus squalene epoxidase gene (ERG1), which encodes the target enzyme of TRB. Authentic A. fumigatus ERG1, amplified from the genome and cloned into pRG3-AMA1-NotI, also conferred TRB-specific resistance. This molecular approach has the potential to enhance our knowledge of the mechanisms of A. fumigatus resistance to modern antifungals.
authors.affiliation: Department of Infectious Diseases, Infection Control and Employee Health, Unit 402, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Gregory S
authors.initials: GS
authors.lastname: May
authors.forename: Michail S
authors.initials: MS
authors.lastname: Lionakis
authors.forename: Russell E
authors.initials: RE
authors.lastname: Lewis
authors.forename: Dimitrios P
authors.initials: DP
authors.lastname: Kontoyiannis
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular; D003470:Culture Media; D053938:DNA Fragmentation / Q000235:genetics; D025141:Drug Resistance, Fungal; D018628:Gene Dosage; D015723:Gene Library; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology*; D010105:Oxygenases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011523:Protoplasts; D050603:Squalene Monooxygenase; D000077291:Terbinafine; D014170:Transformation, Genetic
chemical_list: D000935:Antifungal Agents; D003470:Culture Media; D009281:Naphthalenes; D010105:Oxygenases; D050603:Squalene Monooxygenase; D000077291:Terbinafine