A Candida glabrata erg1 (Cgerg1) mutant, CgTn201S, was identified by transposon mutagenesis and by increased fluconazole susceptibility. CgERG1 encodes a 489-amino-acid protein which, on the basis of its homology with Saccharomyces cerevisiae ERG1, is a squalene epoxidase essential for ergosterol synthesis. Interruption following codon 475 of CgErg1p decreased the ergosterol content by 50%; caused accumulation of the squalene precursor; increased the levels of susceptibility to fluconazole, itraconazole, and terbinafine; increased the level of resistance to amphotericin B; increased the levels of rhodamine 6G and [ 3 H]-fluconazole uptake; reduced the level of growth; and blocked growth under conditions of low oxygen tension. In addition, CgTn201S efficiently took up exogenous cholesterol from cholesterol-containing serum. Cholesterol constituted 34% of the extractable sterols in CgTn201S when it was grown aerobically on serum-containing medium. Under the same conditions, C. albicans contained only 0.1 to 1.2% cholesterol. Exogenous sterols also restored growth under conditions of low oxygen tension. Finally, complementation of the Cgerg1 mutation restored the levels of [ 3 H]fluconazole uptake and drug susceptibility to wild-type levels.Transposon-based insertion into Candida glabrata has been used effectively for mutational analysis (3). We adapted a commercial Tn5-based system for insertion of Tn5 into C. glabrata genomic DNA. After rescue cloning of the Tn5 DNA from the C. glabrata transformants with increased fluconazole susceptibilities, we found that one of the fluconazole-susceptible transformants, CgTn201S, had an insertion in the 3Ј end of the C. glabrata ERG1 (CgERG1) open reading frame (ORF). We explored the mechanism by which this mutation causes fluconazole susceptibility because of our interest in azole resistance in this species. Prolonged treatment of patients with fluconazole causes substantial increases in the levels of azole resistance in C. glabrata. This species is naturally about 8-fold more resistant to fluconazole than Candida albicans, and following drug exposure, the levels of resistance can increase 16-fold more, leading to clinically significant fluconazole resistance (1, 18). The major mechanisms for clinical resistance described to date have been increased drug efflux and alteration of the azole-binding site, the C-14 sterol demethylase, coded for by ERG11. Recently, an erg1 C1228G Saccharomyces cerevisiae mutant was reported to be aerobically viable and to have at least a 10-fold increase in the level of terbinafine resistance but no change in the level of itraconazole resistance (9). Null erg1 mutants of S. cerevisiae are not aerobically viable but are anaerobically viable in the presence of exogenous ergosterol (11). In the absence of data about Cgerg1 mutants, we studied the phenotype of strain CgTn201S, including its azole susceptibility, its ability to grow under conditions of low oxygen tension, and its uptake of exogenous sterols. MATERIALS AND METHODSStrains and culture co...

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Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochromemediated reactions, aerobically synthesized heme. Cytochrome b 5 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.