Genetic analysis of azole resistance in the Darlington strain of Candida albicans

Abstract

High-level azole resistance in the Darlington strain of Candida albicans was investigated by gene replacement in C. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C(14)alpha-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

FIG. 1

(A) Southern analysis for the Darlington strain (lanes 1), the CAI4 strain (lanes 2), and transformant 12 (lanes 3). DNA was digested with the indicated enzymes and was probed with the 0.5-kb ERG11 probe for BsrI, SpeI, PstI, and SpeI plus RcaI. The Southern blot of the HindIII digest was probed with the 1.58-kb ERG11 fragment. (B) Restriction maps of the Darlington and CAI4 strains as well as transformant 12. The following abbreviations indicate restriction sites: B, BsrI; S, SpeI; P, PstI, and R and R′, RcaI. Small boxes indicate the locations of the sequences that hybridized with the probe. The large boxes labeled D-ERG11 and C-ERG11 indicate the locations of the ERG11 ORFs from the Darlington strain and the CAI4 strain, respectively.

FIG. 2

(A) Diagram of the Darlington strain ERG11 ORF. The primers used for PCR were P1878, P1879, K18, and G04. The product obtained by PCR with primers K18 and G04 was used as a probe for Southern analysis with BsrI. (B) Southern blot of RT-PCR product. Double-stranded cDNA was obtained by RT with poly(A)⁺ RNA of C. albicans as the template, followed by PCR amplification with K18 and G04 as primers. The amplified double-stranded cDNA was digested with BsrI, and Southern analysis was performed with a 0.98-kb PCR product (obtained with primers K18 and G04) as a probe. BsrI-digested amplified cDNA of SC5314 and CAI4 showed a 1.0-kb single band (lanes 1 and 2). The cDNA of Darlington showed a 0.9-kb single band (lane 3). Transformant 12 showed both 1.0- and 0.9-kb bands (lane 4). Numbers on the right are in kilobases.

FIG. 3

Fluconazole growth inhibition in C. albicans at day 4 (A) and day 7 (B). Data are for C. albicans isolates CAI4 (open squares), CAF2-1 (open circles), SC5314 (open triangles), Darlington (filled squares), and transformant 12 (filled triangles). OD₄₅₀, optical density at 450 nm.