Abstract
Invasive aspergillosis is mainly caused by the fungal pathogen Aspergillus fumigatus. This type of infection affects approximately 15% of allogeneic transplant patients, with mortality rates ranging from 40% to 90%. Treatments for aspergillosis rely heavily on triazole drugs but A. fumigatus is intrinsically resistant to fluconazole (FLC). Instead, voriconazole (VCZ) has emerged as the preferred antifungal used to treat Aspergillus infections. Despite widespread clinical application, VCZ primary treatment of invasive aspergillosis results in therapeutic failure for a significant proportion of patients. This scenario poses some important experimental questions. How is intrinsic FLC resistance in A. fumigatus conferred and how does a particular set of mutations confer acquired VCZ resistance at the molecular level? Both problems have been addressed by separately expressing in a Saccharomyces cerevisiae system the two isoforms of A. fumigatus sterol 14α-demethylase (AfCYP51A and AfCYP51B) as recombinant full-length functional enzymes together with the cognate NADPH-cytochrome P450 cognate reductase AfCPRA2, and a sterol C24-methyl-transferase AfERG6 that synthesises eburicol from lanosterol. Replacement of ScErg11 with the AfErg6 gene also excluded interference by the native enzyme in assessment of the activity of AfCYP51 isoforms. A requirement for co-expression of AfERG6 with AfCYP51A and AfCYP51B in S. cerevisiae indicated that AfCYP51 enzymes require eburicol but not lanosterol as a substrate. Single amino acid mutations Y121F, T289A, I301T, and the double mutation Y121F T289A in AfCYP51A were used to investigate the interaction of azole drugs with key amino acid residues in the active site of the enzyme. The susceptibility of the S. cerevisiae host and recombinant strains to azole drugs were assessed using agarose diffusion assays and measurement of minimum inhibitory concentrations (MIC80) in liquid microdilution assays. Affinity purified recombinant AfCYP51A and AfCYP51B proteins were used to investigate Type I binding of substrates and Type II binding of selected azoles. Azole susceptibility measurements confirmed both AfCYP51A and AfCYP51B are functional in the host system and indicated AfCYP51A but not CYP51B confers high-level resistance to FLC. Compared to AfCYP51B and S. cerevisiae ERG11, AfCYP51A provided high level resistance to DCZ and VCZ, weaker resistance to VT-1161, but retained susceptibility to PCZ. Type II binding assays gave affinities consistent with the azole susceptibility experiments. Azole susceptibility measurements also showed AfCYP51A T289A conferred 4-fold and 11-fold greater susceptibility to FLC than AfCYP51A I301 and AfCYP51A, respectively. Homology modelling of AfCYP51A suggests that the proximity and polarity of T289 in helix I is likely to interfere with the binding of FLC in the active site, with I301 is located too distant from FLC to interact with it directly. Azole susceptibility experiments showed that the AfCYP51A Y121F plus T289A mutations were required to confer increased resistance to VCZ. These results are consistent with ScERG11 crystal structures showing a water-mediated hydrogen bond network involving the hydroxyl group of Y140 (structurally aligned with Y121 in AfCYP51A), the heme ring D propionate, and the ligand’s tertiary alcohol group affects the binding of FLC, VCZ, and VT-1161 (but not PCZ). This network was overridden by the effect of T289. Functional expression of AfCYP51A and AfCYP51B in yeast and the modelling of AfCYP51A mutations increase understanding of intrinsic resistance of A. fumigatus to FLC and give insight into acquired resistance to short-tailed azole drugs. The recombinant strains enable systematic investigation of the susceptibility of AfCYP51 isoforms to existing azole drugs and aid the discovery of novel antifungals effective against the major fungal pathogen A. fumigatus.