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Microbiology 145 (1999), 2701-2713
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Microbiology (1999), 145, 2701-2713.
© 1999 Society for General Microbiology

Pathogenicity and Medical Microbiology

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

Patrick Marichal1,5, Luc Koymans2, Staf Willemsens1, Danny Bellens1, Peter Verhasselt3, Walter Luyten4, Marcel Borgers5, Frans C. S. Ramaekers5, Frank C. Odds1 and Hugo Vanden Bossche1

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

Author for correspondence: Patrick Marichal. Tel: +32 14 60 31 97. Fax: +32 14 60 54 03. e-mail: pmaricha{at}janbe.jnj.com

The cytochrome P450 14{alpha}-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 G465S). 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.

Keywords: itraconazole, fluconazole, resistance, Erg11p, modelling

The GenBank accession numbers for the sequences reported in this paper are AF153844–AF153850.




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