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Antimicrobial Agents and Chemotherapy, July 2004, p. 2490-2496, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2490-2496.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Wei Liu,1 Gregory S. May,2 Michail S. Lionakis,1 Russell E. Lewis,1,3 and Dimitrios P. Kontoyiannis1,3*

Departments of Infectious Diseases, Infection Control, and Employee Health,1 Department of Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center,2 College of Pharmacy, University of Houston, Houston, Texas3

Received 10 December 2003/ Returned for modification 14 January 2004/ Accepted 15 March 2004

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 µg/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.

* Corresponding author. Mailing address: 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. Phone: (713) 792-6237. Fax: (713) 745-6839. E-mail: dkontoyi{at}mdanderson.org.

Antimicrobial Agents and Chemotherapy, July 2004, p. 2490-2496, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2490-2496.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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