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 lack of genetics in A. fumigatus, limited 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 multi-copy nonintegrating vector pRG3-AMA1-NotI that also has the pyr-4 gene for selection). We obtained one TRB-resistant pyrG+ transformant in minimal medium minus uracil plates that contained a fungicidal concentration (0.625 μg/ml) of TRB. To determine whether TRB resistance in that transformant was plasmid-dependent, we evicted the plasmid from it by subculturing it on a nutrient-rich medium. We found that loss of the plasmid resulted in loss of both uracil prototrophy and TRB resistance in that transformant. We further identified the gene conferring TRB resistance as the A. fumigatus squalene epoxidase gene, which encodes for the target enzyme of TRB. We then subcloned the complete gene into the vector pRG3-AMA1-NotI and transformed it into A. fumigatus protoplasts. Again, squalene epoxidase, in increased copies, resulted in TRB-specific resistance in A. fumigatus. This molecular approach has the potential to enhance our knowledge of the mechanisms of A. fumigatus resistance to modern antifungals.
Full conference title:
14th Annual Focus on Fungal Infections
- FFI 14th (2004)