[Cloning of the Aspergillus fumigatus squalene epoxidase gene].
OBJECTIVE
To clone Aspergillus fumigatus squalene epoxidase gene and to further investigate its role in terbinafine resistance.


METHODS
The A.fumigatus genomic DNA library was transformed into pyrG-A. fumigatus strain protoplasts with polyethylene glycol-mediated transformation protocol. TRB-resistant pyrG+ transformants were then isolated by being plated on MM-U with TRB (0.625 mg/L) plates. After confirmation of terbinafine-resistance by using both disk diffusion and NCCLS M38-A microdilution antifungal susceptibility testing, the gene conferring terbinafine-resistance was identified. Finally, the gene was cloned and retransformed into pyrG-A. fumigatus strain.


RESULTS
From a total of 5x10(4) transformants, one TRB-resistant pyrG+ transformant was isolated, which showed the terbinafine-specific resistance without cross-resistance to any other antifungals. A. fumigatus squalene epoxidase gene was further identified to confer this terbinafine-resistance. As a result, the complete A. fumigatus squalene epoxidase gene was firstly cloned. Finally, the transformants with extra copies of A. fumigatus squalene epoxidase gene, again, showed the specific resistance to terbinafine.


CONCLUSIONS
Extra copies of A. fumigatus squalene epoxidase gene, which was cloned for the first time in this study, could result in A. fumigatus resistance to terbinafine. This is a novel mechanism of terbinafine-resistance that needs further investigation for its clinical significance.
Liu
Wei
W
Department of Dermatology, Peking University First Hospital, Beijing 100034, China.
May
Gregory S
GS
Kontoyiannis
Dimitrios P
DP
Li
Ruo-yu
RY
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D025141:Drug Resistance, Fungal / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology*; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D050603:Squalene Monooxygenase / Q000235:genetics*; D000077291:Terbinafine; D014170:Transformation, Genetic
D000935:Antifungal Agents; D009281:Naphthalenes; D050603:Squalene Monooxygenase; D000077291:Terbinafine