Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei

Infections with Candida krusei have increased in recent years as a conseque nce of its intrinsic resistance to fluconazole, an antifungal azole widely used in immunocompromised individuals to suppress infections due to azole-s usceptible C. albicans. One established mechanism for azole resistance is d rug efflux by ATP binding cassette (ABC) transporters. Since these transpor ters recognize structurally diverse drugs, their overexpression can lead to multidrug resistance (MDR). To identify C. krusei genes potentially involv ed in azole resistance, PCR was performed with primers corresponding to con served sequences of MDR-related ABC transporters from other fungi. Two gene s, ABC1 and ABC2, were identified; Southern blots suggested that both have one or two related gene copies in the C. krusei genome. ABC1 RNA was consti tutively expressed at low levels in log phase cells while ABC2 RNA was unde tectable. However, both genes were upregulated as cultures approached stati onary phase, and this upregulation was correlated with decreased susceptibi lity to the lethal activity of the azole derivative miconazole. Furthermore , ABC1 was upregulated following brief treatment of C. krusei with miconazo le and clotrimazole (but not other azoles), and the unrelated compounds alb endazole and cycloheximide. The latter two compounds antagonized fluconazol e activity versus C. krusei, supporting a role for the ABC1 transporter in azole efflux. Finally, miconazole-resistant mutants selected in vitro demon strated increased constitutive expression of ABC1. Based on these expressio n data, genetic and functional characterization of the ABC1. transporter to directly test its role in C. krusei azole resistance would appear to be wa rranted.