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

The fungal end product sterol, ergosterol, and its biosynthetic pathway are the targets of the majority of the antifungal compounds currently in use for human infections and agricultural applications. The primary class of compounds used in human infections is the azoles, drugs that inhibit the C-14 demethylation of the pathway intermediate lanosterol. Overuse of the azoles, especially in immunocompromised patients, has led to increases in the incidence of antifungal resistance (5). As resistance levels to these compounds rise, the need to identify new targets for antifungal therapy expands. Several reactions in the ergosterol biosynthetic pathway of the pathogenic fungus Candida albicans have been identified (1, 18, 26) as required for viability and, thus, might be subject to exploration as new antifungal sites. Inhibition of these steps could prove to be effective antifungal interventions because this organism cannot import exogenous sterol (19, 29), making sterol biosynthesis obligatory. Sterol biosynthesis in fungi is an exclusively aerobic process because several steps in the ergosterol biosynthetic pathway require molecular oxygen or heme, which are also synthesized only under aerobic conditions and used primarily in cytochrome-mediated reactions. Among the latter are the cytochrome P-450-dependent step in lanosterol demethylation (encoded by CYP51, also referred to as ERG11 and ERG16), the cytochrome P-450-dependent desaturation step at C-22 (encoded by CYP61, also referred to as ERG5), and the cytochrome b5 requiring C-5 desaturation (encoded by ERG3). In the case of the cytochrome P-450 steps, two distinct enzymes are involved (11, 12), but each requires the same electron donor, the NADPH cytochrome P-450 oxidoreductase (encoded by NCP1). Ncp1p also donates electrons in the presterol, oxygen-requiring reaction catalyzed by squalene epoxidase (32). The cytochrome b5 step in the pathway utilizes NADH-cytochrome b5 reductase or NADPH-cytochrome c reductase as electron carriers in the creation of the double bond at C5-6 (25). Sutter and Loper (27) reported that disruption of the Saccharomyces cerevisiae NCP1 gene is not lethal despite the fact that two steps in the pathway utilize this electron donor and that one of the steps, the C-14 demethylation of lanosterol, has proven to be essential based on the fact that disruption of the cytochrome P-450 demethylase gene (CYP51) results in nonviability (17). The presence of an alternative electron carrier, perhaps cytochrome b5, was postulated to explain the viability of the ncp1 mutant (13). This substitution of function by cytochrome b5 was confirmed in a follow-up study (20). Similarly, the gene encoding cytochrome b5 in S. cerevisiae was found to be nonessential (28). Disruption of CYB5 generates no growth phenotype in a wild-type background but results in lethality when present in a ncp1 background (28). This suggests that NCP1 might provide a reciprocal function for the missing cytochrome b5 protein. Ncp1p and Cyb5p appear to have overlapping functions in S. cerevisiae. One of the phenotypes of ncp1 is an increased sensitivity to azole antifungals (27). This is postulated to be due to the increased sensitivity of the substitute electron carrier in the C-14 demethylation reaction and suggests a mechanism of action for azoles beyond interacting with Cyp51p. A more recent study (28) has confirmed the azole sensitivity of the ncp1 phenotype and has demonstrated that ncp1 cells still produce about 25% of the ergosterol produced by NCP1 strains. In addition, elevated levels of lanosterol were not detected, indicating that the cells were able to complete the C-14 demethylation reaction as well as the other reactions where Ncp1p normally functions. Based on an in vitro assay (13) and on suppression of the ncp1 phenotype by CYB5 (28), Cyb5p could be the replacement electron donor. Based on the increased azole sensitivity of ncp1 strains coupled with the fact that required reactions in sterol biosynthesis in S. cerevisiae and C. albicans can differ (16), it is worthwhile to investigate the characteristics of the cyb5 and the ncp1 phenotypes in this pathogenic fungus. This report investigated whether CYB5 is an essential gene and whether Cyb5p may be a potential drug target. This report also employed various methods of gene disruption due to difficulties in isolating homozygous mutants for the determination of gene essentiality.