Inhibitor Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition.

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated twenty Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (eight mutants), enniatin B (eight) or beauvericin (four). The twenty mutations were in just nine residues at the centres of transmembrane segment 1 (TMS1) (six mutations), TMS4 (four), TMS5 (four), TMS8 (one) and TMS11 (two) and in A713P (three), a previously reported FK506-resistant 'hotspot 1' mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) isolates were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25 resistant mutants were altered in four amino acids: G521R, M639I, A713P and T1355N. These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N (TMS11) possibly cause inhibitor-resistance by altering TMS-contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.