Complementation of a Saccharomyces cerevisiae ERG11/CYP51 (sterol 14α-demethylase) doxycycline-regulated mutant and screening of the azole sensitivity of Aspergillus fumigatus isoenzymes CYP51A and CYP51B.
Aspergillus fumigatus sterol 14α-demethylase isoenzymes CYP51A and CYP51B were heterologously expressed in a Saccharomyces cerevisiae mutant (YUG37-erg11), wherein native ERG11/CYP51 expression is controlled using a doxycycline-regulatable promoter. When cultured in the presence of doxycycline, recombinant YUG37-pcyp51A and YUG37-pcyp51B yeasts were able to synthesize ergosterol and grow; a control strain harboring reverse-oriented cyp51A could not. YUG37-pcyp51A and YUG37-pcyp51B constructs showed identical sensitivity to itraconazole, posaconazole, clotrimazole, and voriconazole. Conversely, YUG37-pcyp51A withstood 16-fold-higher concentrations of fluconazole than YUG37-pcyp51B (8 and 0.5 μg ml⁻¹, respectively).
Martel
Claire M
CM
Institute of Life Science and School of Medicine, Swansea University, Swansea, Wales, United Kingdom.
Parker
Josie E
JE
Warrilow
Andrew G S
AG
Rolley
Nicola J
NJ
Kelly
Steven L
SL
Kelly
Diane E
DE
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology*; D001393:Azoles / Q000494:pharmacology*; D003022:Clotrimazole / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology*; D004875:Ergosterol / Q000378:metabolism; D015725:Fluconazole / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D017964:Itraconazole / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014230:Triazoles / Q000494:pharmacology; D065819:Voriconazole
D000900:Anti-Bacterial Agents; D001393:Azoles; D005656:Fungal Proteins; D011743:Pyrimidines; D029701:Saccharomyces cerevisiae Proteins; D014230:Triazoles; D017964:Itraconazole; C101425:posaconazole; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus; C465527:cytochrome P-450 CYP51B, Aspergillus; C526348:Erg11 protein, S cerevisiae; D003022:Clotrimazole; D065819:Voriconazole; D004318:Doxycycline; D004875:Ergosterol