title: Genotype-phenotype complexity of the TR46/Y121F/T289A cyp51A azole resistance mechanism in Aspergillus fumigatus.
abstract: The Aspergillus fumigatus cyp51A gene TR46/Y121F/T289A mutation is a new emerging resistance mechanism with high-level voriconazole (VOR) resistance, and elevated MICs to all other medical azoles. This is highly worrisome as VOR is the primary drug for the treatment of many aspergillus diseases. The 46 base pair tandem repeat (TR46) is positioned at the same location of the cyp51A gene promoter region as has been described for other tandem repeats. The exact role of the TR46 in combination with the two amino acid changes (Y121F and T289A) in the CYP51A protein is unknown. In this study this azole resistance mechanism was investigated by recombinant analysis study combined with homology modelling. MICs of the TR46/Y121F/T289A recombinant corresponded to the MICs of the original clinical isolates containing the same mutations with high-level resistance to VOR. The TR46 or Y121F by itself has only a moderate effect on azole susceptibility. The combination of TR46/Y121F, however, appears to be highly resistant not only for VOR but also for itraconazole (ITZ). The genetic change of T289A in combination with TR46 or by itself has no significant effect on the phenotype but moderates the phenotype of the ITZ resistance only in the presence of Y121F. The striking resistant phenotype of the TR46/Y121F mutant is supported by the structural analysis of the CYP51A homology model. The A. fumigatus CYP51A Y121 residue forms an H-bond with the heme centre of the enzyme. Disruption of the H-bond by the Y121F substitution destabilizes the active centre of CYP51A which appears to be essential with respect to azole resistance. In CYP51A-azole complexes, residue T289 is in close proximity of the azole moiety of VOR. Replacement of the polar amino acid threonine by the more hydrophobic amino acid alanine might promote more stable drug-protein interactions and has thereby an impact on ITZ susceptibility, which is confirmed by the MICs of the genetic recombinants.
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: Eveline
authors.initials: E
authors.lastname: Snelders
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: Simone M T
authors.initials: SM
authors.lastname: Camps
authors.affiliation: Medicinal Chemistry, Taros Chemicals GmbH, Emil-Figge-Str. 76a, Dortmund, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Karawajczyk
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: Antonius J M M
authors.initials: AJ
authors.lastname: Rijs
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Zoll
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Verweij
authors.affiliation: Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands. Electronic address: Willem.Melchers@radboudumc.nl.
authors.forename: Willem J G
authors.initials: WJ
authors.lastname: Melchers
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000494:pharmacology; D001232:Aspergillus fumigatus / Q000187:drug effects* / Q000235:genetics*; D001393:Azoles / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D056726:Genetic Association Studies; D005838:Genotype; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D005656:Fungal Proteins; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus
keywords: Aspergillus fumigatus; Homology modelling; Multi-azole resistance; cyp51A