Molecular analysis of Candida albicans ABC transporter Cdr1p

Resistance of Candida albicans to widely used azoles poses serious clinical problems, as C. albicans is the predominant cause of systemic fungal infections in immunocompromised individuals. The major mechanism of azole resistance is the overexpression of ATP-binding cassette (ABC) transporters such as C. albicans Cdr1p. A detailed analysis of 244 fungal pleiotropic drug resistance (PDR) type ABC transporters revealed highly conserved amino acid motifs in the two large fungal PDR transporter-specific extracellular loops (EL3 and EL6) that are essential for its function and localisation in the plasma membrane. Previous studies identified E704 in EL3 as a critically important amino acid for proper folding and localisation of Cdr1p. Objective: This project investigated the hypothesis that the conserved amino acid R546 interacts with E704 in Cdr1p and compensates for the loss of function caused by the E704K mutation.  Methods:  The R546E mutation was introduced into wild-type Cdr1p to make a single mutant, and into previously constructed EL3.1 mutant (E704K) to make a double mutant using PCR-based site-directed mutagenesis and transformation of Saccharomyces cerevisiae ADD. Phenotypes of the mutants were analysed using drug susceptibility and pump chemosensitisation assays. Results:   Changing R546 to E546 had no effect on the transport function of Cdr1p. Cdr1p-R546E behaved similarly to the wild-type enzyme and responded equally well to known Cdr1p efflux pump inhibitors, although the double mutant R546E/E704K became slightly more susceptible to test drugs than the parent EL3.1. Conclusions: R546 does not appear to interact with E704. Also, it was clear that R546 is not in close contact with E704, disproving the hypothesis that R546 creates a salt-bridge with E704 that is important for the proper folding and function of the enzyme. Nevertheless, the single ADD/CDR1A-R546E and double ADD/CDR1A-R546E.E704K mutants are useful for further analysis of Cdr1p function.