title: Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14alpha-demethylase.
abstract: To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10(8) and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [(14)C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14alpha-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.
authors.affiliation: Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. paul.mcnicholas@spcorp.com
authors.forename: Paul A
authors.initials: PA
authors.lastname: Mann
authors.forename: Raulo M
authors.initials: RM
authors.lastname: Parmegiani
authors.forename: Shui-Qing
authors.initials: SQ
authors.lastname: Wei
authors.forename: Cara A
authors.initials: CA
authors.lastname: Mendrick
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: David
authors.initials: D
authors.lastname: Loebenberg
authors.forename: Beth
authors.initials: B
authors.lastname: DiDomenico
authors.forename: Roberta S
authors.initials: RS
authors.lastname: Hare
authors.forename: Scott S
authors.initials: SS
authors.lastname: Walker
authors.forename: Paul M
authors.initials: PM
authors.lastname: McNicholas
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D010088:Oxidoreductases / Q000235:genetics*; D017354:Point Mutation; D058886:Sterol 14-Demethylase; D014230:Triazoles / Q000494:pharmacology*
chemical_list: D000935:Antifungal Agents; D014230:Triazoles; C101425:posaconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase