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Antimicrobial Agents and Chemotherapy, September 2004, p. 3425-3435, Vol. 48, No. 9
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.9.3425-3435.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Disruption of the Candida albicans CYB5 Gene Results in Increased Azole Sensitivity

Department of Biology, Indiana University Purdue University Indianapolis,¹ Department of Drug Disposition, Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana²

Received 31 December 2003/ Returned for modification 29 January 2004/ Accepted 18 May 2004

Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme. Cytochrome b₅ is required for sterol C5-6 desaturation and the encoding gene, CYB5, is nonessential in Saccharomyces cerevisiae. Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable. The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals. Based on this phenotype, the CYB5 gene in the human pathogen Candida albicans was investigated to determine whether the cyb5 genotype was viable and would also demonstrate azole sensitivity. Sequential disruption of the CYB5 alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the CYB5 gene. Subsequent disruption procedures with a pMAL2-CYB5 rescue cassette and a CYB5-URA3 blaster cassette resulted in viable cyb5 strains with no third copy. The C. albicans CYB5 gene is concluded to be nonessential. Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol. The C. albicans cyb5 strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the S. cerevisiae cyb5. The C. albicans cyb5 shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the ERG2 and ERG24 gene products. These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment.

M.B. and N.D.L. dedicate this manuscript to the memory of their mothers, Tillie Bard and Adeline Lees.