Contribution of mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans

Microbiology (Reading). 1999 Oct;145 ( Pt 10):2701-2713. doi: 10.1099/00221287-145-10-2701.

Abstract

The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Antifungal Agents / metabolism
  • Antifungal Agents / pharmacology*
  • Candida albicans / drug effects*
  • Candida albicans / enzymology
  • Candida albicans / genetics*
  • Chromosome Mapping
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / genetics*
  • Cytochrome P-450 Enzyme System / metabolism
  • Drug Resistance, Microbial
  • Drug Resistance, Multiple
  • Fluconazole / metabolism
  • Fluconazole / pharmacology
  • Itraconazole / metabolism
  • Itraconazole / pharmacology
  • Microbial Sensitivity Tests
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Oxidoreductases / chemistry
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Protein Conformation
  • Protein Structure, Secondary
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Sterol 14-Demethylase

Substances

  • Antifungal Agents
  • Itraconazole
  • Fluconazole
  • Cytochrome P-450 Enzyme System
  • Oxidoreductases
  • Sterol 14-Demethylase

Associated data

  • GENBANK/AF153844
  • GENBANK/AF153845
  • GENBANK/AF153846
  • GENBANK/AF153847
  • GENBANK/AF153848
  • GENBANK/AF153849
  • GENBANK/AF153850