title: Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2.
abstract: Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.
authors.affiliation: Department of Oral Sciences, School of Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: Ann R
authors.initials: AR
authors.lastname: Holmes
authors.forename: Sarah
authors.initials: S
authors.lastname: Tsao
authors.forename: Soo-Wee
authors.initials: SW
authors.lastname: Ong
authors.forename: Erwin
authors.initials: E
authors.lastname: Lamping
authors.forename: Kyoko
authors.initials: K
authors.lastname: Niimi
authors.forename: Brian C
authors.initials: BC
authors.lastname: Monk
authors.forename: Masakazu
authors.initials: M
authors.lastname: Niimi
authors.forename: Aki
authors.initials: A
authors.lastname: Kaneko
authors.forename: Barbara R
authors.initials: BR
authors.lastname: Holland
authors.forename: Jan
authors.initials: J
authors.lastname: Schmid
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cannon
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000502:physiology; D000483:Alleles; D000935:Antifungal Agents / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D015153:Blotting, Western; D002176:Candida albicans / Q000187:drug effects / Q000235:genetics*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D015725:Fluconazole / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics; D006579:Heterozygote; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000502:physiology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D001393:Azoles; C094693:CDR1 protein, Candida albicans; D004271:DNA, Fungal; D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D015725:Fluconazole