Amino acid residues critical for drug pump function in Candida albicans

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The most common cause of high-level azole resistance in clinical isolates of Candida albicans is over-expression of drug efflux proteins, in particular, the ABC-type membrane transporters Cdr1p and Cdr2p. We examined the function of individual efflux proteins by expressing them in the genetically tractable yeast Saccharomyces cerevisiae. Individual CDR1 and CDR2 alleles were cloned and hyper-expressed in an S. cerevisiae expression system (host strain AD1-8u^- and transformation cassette constructed in plasmid pABC3) that correctly trafficked the hyper-expressed efflux pump protein to the host yeast plasma membrane.
Recombinant S. cerevisiae strains were constructed that expressed variants of one allele (A) of the C. albicans ATCC 10261 CDR1 gene generated by low-fidelity PCR mutagenesis. Specific regions of the CDR1 gene, such as the Nucleotide Binding Domain (NBD) were deleted and replaced by mutagenised PCR-generated fragments. One recombinant had a reduced fluconazole Minimum Inhibitory Concentration (MIC) of 75 μg/ml relative to the parental A allele strain (200 μg/ml), reduced rhodamine 6G (R6G) efflux, and contained two unique amino acid substitutions (F371S and R420S) in NBD1. Comparative SDS-PAGE analysis demonstrated reduced amounts of Cdr1p in the plasma membrane of this strain, possibly due to instability of the mutant polypeptide. In another mutant CDR1 allele strain, a L1021S mutation, immediately N-terminal of the NBD2 Walker B motif, conferred reduced pump activity without reducing Cdr1p expression in the plasma membrane. The hyper-expression system was also used to investigate natural allelic variation in both CDR1 and CDR2 in eight strains. Whereas only one strain had non-synonymous single nucleotide polymorphisms (SNPs) within the CDR1 gene, that gave minor functional change, the CDR2 alleles of seven strains contained both intra- and inter-strain non-synonymous SNPs. Functional variation between CDR2 alleles was detected, and confirmed by site-directed mutagenesis of individual SNPs.
The use of a heterologous hyper-expression system has facilitated analysis of allelic variation and identified amino acid residues important for function in the C. albicans drug efflux genes CDR1 and CDR2.