Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resistance to azole antifungals in Candida glabrata

Candida glabrata has been often isolated from AIDS patients with oropharyng eal candidiasis treated with azole antifungal agents, especially fluconazol e. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDRI was upregulated in C.glabrata clinical isolates resistant to azole a ntifungal agents (D. Sanglard, F, Ischer, D, Calabrese, P, A. Majcherczyk, and J, Bille, Antimicrob, Agents Chemother, 43:2753-2765, 1999), Deletion o f CgCDRI in C.glabrata rendered the null mutant hypersusceptible to azole d erivatives and showed the importance of this gene in mediating azole resist ance. We observed that wild-type C,glabrata exposed to fluconazole in a med ium containing the drug at 50 mug/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 x 10(-4) to 4 x 10(-4)). We show here that this high-frequency azole resistance (HFAR) acquired in v itro was due, at least in part, to the upregulation of CgCDRI, The CgCDRI d eletion mutant DSY1014 could still develop HFAR but in a medium containing fluconazole at 5 mug/ml. In the HFAR strain derived from DSY1041, a distinc t ABC transporter gene similar to CgCDRI, called CgCDR2, was upregulated, T his gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype, Deletion of both CgCDRI and CgCDR2 suppre ssed the development of HFAR in a medium containing fluconazole at 5 mug/ml , showing that both genes are important mediators of resistance to atole de rivatives in C.glabrata. We also show here that the HFAR phenomenon was lin ked to the loss of mitochondria in C.glabrata, Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to atole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitocho ndrial loss was at least linked to the upregulation of both CgCDRI and CgCD R2.