Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14␣-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.Candida krusei is an opportunistic pathogen that can cause serious infections in immunocompromised patients (1, 6, 7). This organism is intrinsically resistant to fluconazole. The new triazole voriconazole has greater in vitro activity than fluconazole against C. krusei (5, 15). Two mechanisms of azole resistance in C. krusei have been described. Isolates of C. krusei that are resistant to itraconazole exhibit reduced drug accumulation, suggesting that resistance to this drug is due to the activity of one or more drug efflux pumps (29). Recently, two ATP binding cassette transporters have been identified in C. krusei. Increased expression of these transporters is associated with reduced susceptibility to miconazole (9).A second mechanism of azole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14␣-demethylase (CYP51), to inhibition by an azole antifungal agent. We have determined previously that fluconazole resistance in some strains of C. krusei is mediated predominantly by this mechanism (17).In the present study, we cloned the full-length C. krusei CYP51 and examined its contribution to the differential sensitivity of C. krusei to fluconazole and voriconazole. We also used computer-assisted molecular modeling to examine the interactions of fluconazole and voriconazole with the predicted Candida albicans and C. krusei CYP51s. MATERIALS AND ME...
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