Functional Analysis of Phosphorylation Sites in Candida albicans Cdr1p

Fluconazole (FLC) has become the antifungal drug of choice for non-life-threatening Candida infections. However, there is an increasing clinical problem of drug resistance. This frequently results from elevated expression of the Candida albicans ATP-binding cassette (ABC) drug efflux pumps, such as Cdr1p and Cdr2p, in the yeast plasma membrane. Phosphorylation is a critical element of ABC pump activity. Objectives: To examine the function of two putative phosphorylation sites in Cdr1p, the major C. albicans drug efflux pump, by undertaking site-directed mutagenesis (SDM) and expression of mutant alleles in the heterologous host, Saccharomyces cerevisiae. Methods: SDM was accomplished by recombinant PCR in which the serines at the putative phosphorylation sites S312 and S487 were replaced with either alanine or aspartate. The template for the recombinant PCRs was a plasmid containing the CDR1B allele of C. albicans ATCC 10261. Cassettes were constructed comprising the mutant CDR1 gene, a selection marker (URA3) and flanking sequences of the S. cerevisiae PDR5 gene. These cassettes were used to transform the heterologous host strain S. cerevisiae (AD delta) to ura+. The effects of the introduced mutations were determined using functional assays of fluconazole (FLC) susceptibility, and rhodamine 6G (R6G) efflux. Results: The S487A(D) mutants had FLC susceptibilities and R6G efflux activities similar to the strain expressing the parental CDR1 allele. The S312A(D) mutants showed increased susceptibilities to FLC and decreased R6G efflux. A strain containing both the S312A and the S487A mutations had similar properties to the S312A mutant whereas the double S312D/S487D mutant was the most affected with the least resistance to FLC and reduced R6G efflux activity. Conclusion: Mutation of the phosphorylation site S312, or double mutation of S312 and S487, reduced Cdr1p function, whereas S487 mutations had negligible effects, demonstrating that S312 is a major C. albicans phosphorylation site.