Aneuploidy and isochromosome formation in drug-resistant Candida albicans

Abstract

Resistance to the limited number of available antifungal drugs is a serious problem in the treatment of Candida albicans. We found that aneuploidy in general and a specific segmental aneuploidy, consisting of an isochromosome composed of the two left arms of chromosome 5, were associated with azole resistance. The isochromosome forms around a single centromere flanked by an inverted repeat and was found as an independent chromosome or fused at the telomere to a full-length homolog of chromosome 5. Increases and decreases in drug resistance were strongly associated with gain and loss of this isochromosome, which bears genes expressing the enzyme in the ergosterol pathway targeted by azole drugs, efflux pumps, and a transcription factor that positively regulates a subset of efflux pump genes.

Fig. 1

A common segmental aneuploidy appears in Flu^R strains. (A) Aneuploidy was common in Flu^R strains (table S1). Number of whole-chromosome aneuploidies in Flu^S (white) and Flu^R (black) and number of segmental aneuploidies, seen only in Flu^R strains (gray). (B) A common chr5 breakpoint in Flu^R strains. Panels 1 through 8 represent CGH plots of chr5 for YJB8735, YJB9185, YJB8640, YJB9311, YJB8738, YJB8736, YJB8737, and YJB9180. The y axis plots gene copy number calculated from log 2 values. Two shorter segmental aneuploidies are seen in panel 8. Diagram illustrates the fine structure of the breakpoint including the Cse4p site and the two insertions found on one homolog. IR, inverted repeat; MTL, mating type–like. (C) Increased gene copy number correlates with increased gene expression. Transcript profile data (mean log 2 values) for strain YJB9311 () were plotted by using a running average over five ORFs. (D) CGH data of chr5 from a Flu^S strain reveals an i(5R) with the same breakpoint as strains with i(5L).

Fig. 2

The segmental aneuploidies on chr5 reflect isochromosome structures. (A) All strains from Fig. 1B contain a unique restriction fragment that spans the Cse4p binding site (lanes 2 to 5, 9, and 11 to 13). Southern blot of Eco NI–digested genomic DNA probed with Cse4p binding site sequence (black bar) detects an ~10-kbp fragment diagnostic of i(5L) structure. Differences in the size of this fragment are due to polymorphic insertion sequences (Fig. 1B). WT: wild type (SC5314), lanes 1 to 15: YJB8734, YJB8735, YJB8736, YJB8737, YJB8738, YJB8739, YJB8740, YJB9309, YJB9311, YJB9175, YJB9180, YJB9185, YJB8640, YJB9613, and YJB8638. Lane designations are identical for all other parts of this figure. (B to H) The isochromosome is either independent or attached to a homolog of chr5. Whole-chromosome CHEF gels stained with ethidum bromide (B), blotted, and probed with chr5L (C) or chr5R (D) reveal an independent isochromosome (lanes 2 to 4, 9, and 12) or an attached isochromosome (lanes 5 and 11). Sfi I digestion of whole chromosomes separated by CHEF and stained with ethidium bromide (E); blotted; and probed with chr5L (F), chr5R (G), and chr5I (H) probes [diagramed in (J, K, and L)] show that the independent isochromosome (lanes 9, 12, and 13) does not contain an Sfi I site. Sfi I digestion of the attached isochromosome (lane 11) releases fragments expected if i(5L) were attached to the left arm of whole chr5. (I) The attached isochromosome includes a telomere-telomere junction. Southern blot analysis of Sac I–digested genomic DNA probed with a chr5L telomere-adjacent probe detected a 10-kbp telomere fragment in all wild-type (WT) and parental (P) strains and an additional larger fragment in YJB8738 (lane 5), as would be expected if two chr5L arms attached via telomere sequence (L). YJB9180 contains a complex attachment not including this Sac I fragment. (J to L) Diagrams, including relevant restriction sites and probes: (J) normal chr5, (K) independent i(5L), and (L) attached i(5L). The centromere region (inverted repeat and central unique sequence) is not to scale.

Fig. 3

Loss of i(5L) is associated with a dramatic decrease in Flu^R. Whole-genome CGH plots reveal, compared with (A), the gain (B) and loss (C) of i(5L) (left panels) and fluconazole-resistance by E-test assay (right panels) in isolates: (A) YJB8638, MIC = 2.0 μg/ml; YJB9185, (B) MIC > 256 μg/ml; (C) YJB9517, MIC = 8 μg/ml.