Contribution of mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans.
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.
Marichal
Patrick
P
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.|Department of Molecular Cell Biology and Genetics, University of Maastricht, The Netherlands5.
Koymans
Luc
L
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.
Willemsens
Staf
S
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.
Bellens
Danny
D
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.
Verhasselt
Peter
P
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.
Luyten
Walter
W
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.
Borgers
Marcel
M
Department of Molecular Cell Biology and Genetics, University of Maastricht, The Netherlands5.
Ramaekers
Frans C S
FCS
Department of Molecular Cell Biology and Genetics, University of Maastricht, The Netherlands5.
Odds
Frank C
FC
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.
Vanden Bossche
Hugo
H
Department of Anti-infectives Research1, Center for Molecular Design2, Department of Biotechnology3 and Department of Functional Genomics4, Janssen Research Foundation, Turnhoutseweg 30, B2340 Beerse, Belgium.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology*; D002176:Candida albicans / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D002874:Chromosome Mapping; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D015725:Fluconazole / Q000378:metabolism / Q000494:pharmacology; D017964:Itraconazole / Q000378:metabolism / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D058886:Sterol 14-Demethylase
D000935:Antifungal Agents; D017964:Itraconazole; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase