TY - JOUR
T1 - Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine
T2 - Genetic approach to studying gene dose-dependent resistance to antifungals in A. fumigatus
AU - Liu, Wei
AU - May, Gregory S.
AU - Lionakis, Michail S.
AU - Lewis, Russell E.
AU - Kontoyiannis, Dimitrios P.
PY - 2004/7
Y1 - 2004/7
N2 - 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.
AB - 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.
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U2 - 10.1128/AAC.48.7.2490-2496.2004
DO - 10.1128/AAC.48.7.2490-2496.2004
M3 - Article
C2 - 15215099
AN - SCOPUS:3042678033
SN - 0066-4804
VL - 48
SP - 2490
EP - 2496
JO - Antimicrobial agents and chemotherapy
JF - Antimicrobial agents and chemotherapy
IS - 7
ER -