Genomic plasticity of the human fungal pathogen Candida albicans

Abstract

The genomic plasticity of Candida albicans, a commensal and common opportunistic fungal pathogen, continues to reveal unexpected surprises. Once thought to be asexual, we now know that the organism can generate genetic diversity through several mechanisms, including mating between cells of the opposite or of the same mating type and by a parasexual reduction in chromosome number that can be accompanied by recombination events (2, 12, 14, 53, 77, 115). In addition, dramatic genome changes can appear quite rapidly in mitotic cells propagated in vitro as well as in vivo. The detection of aneuploidy in other fungal pathogens isolated directly from patients (145) and from environmental samples (71) suggests that variations in chromosome organization and copy number are a common mechanism used by pathogenic fungi to rapidly generate diversity in response to stressful growth conditions, including, but not limited to, antifungal drug exposure. Since cancer cells often become polyploid and/or aneuploid, some of the lessons learned from studies of genome plasticity in C. albicans may provide important insights into how these processes occur in higher-eukaryotic cells exposed to stresses such as anticancer drugs.

Fig. 1.

CHEF karyotype gel electrophoresis of C. albicans clinical isolates. CHEF gels were run with optimal separation of Chr2 to Chr7 and then stained with ethidium bromide. (A) A clinical isolate with a standard karyotype; (B) different clinical isolates with nonstandard karyotypes, including additional bands due to differences in size between homolog pairs and/or possible chromosome translocations. (C) Schematic diagram of the MRS on Chr5 (106); XhoI cuts outside the MRS, while there are multiple SfiI cut sites within each RPS subunit. The entire MRS on Chr5 ranges from 16 to 92 kb in size (106) (diagram is not to scale; HOK, ∼8kb; RB2, ∼6kb; RPS, ∼2kb each).

Fig. 2.

Mechanisms that can result in LOH. (A) LOH due to mitotic recombination: short-range LOH can arise from gene conversion between homologous chromosomes (Hom a and Hom b) or from double crossovers (XO); long-range LOH events can arise from single crossovers followed by mitotic segregation or from break-induced replication (BIR). (B) Events due to chromosome missegregation: chromosome nondisjunction yields aneuploid cells monosomic for that chromosome (b) as well as cells carrying an extra copy of the homolog (aab). Events due to reduplication of the monosomic chromosome via a subsequent nondisjunction event or by unlicensed rereplication can generate a strain that is homozygous but disomic for that chromosome (bb).

Fig. 3.

Complementary methods used to detect genome changes in four related isolates (derived from clinical isolate T118) that evolved fluconazole resistance in vitro (41, 175). (A) CHEF karyotype gel does not detect differences between the strains; (B) a Southern blot of this CHEF gel, probed with CEN5 DNA, reveals the presence of a SNC in three of the four isolates. This SNC is i(5L), which is similar in size to Chr7 and carries two genes that confer Flu^R in a copy number-dependent manner (174); (C) aCGH detects the i(5L) in these three isolates and also reveals additional whole-chromosome and segmental aneuploidies as noted to the right of each aCGH plot; (D) flow cytometry reveals major shifts in ploidy to approximately three whole-genome copies (∼3C, red) or four copies (∼4C, green). In contrast, no genomic changes other than SNPs are detected in isolate D8-330 (37, 175). (E) ERG11 and TAC1, two well-characterized genes involved in fluconazole resistance, are both found on the left arm of Chr5 (Chr5L), ∼148 kb from the telomere and ∼14 kb to the right of MTL, respectively. (F) Because all i(5L) SNCs analyzed carry homozygous copies of genes on both Chr5L arms (; A. Selmecki and J. Berman, unpublished data), we propose that the isochromosome forms via a break-induced replication involving the inverted repeat that surrounds CEN5. Flow cytometry data have not been published previously.

Fig. 4.

Genome rearrangements mediated by translocations at MRS sequences in C. albicans isolates. (A to C) CHEF karyotype diagrams of strains 1006, WO-1, and WO-2 (118). WO-1 underwent three reciprocal translocation events, resulting in 6 new fusion chromosomes. All translocations occurred at or near the MRS, resulting in identical SfiI digestion patterns between 1006 and WO-1 (35). WO-2 (derived from WO-1) underwent multiple chromosome nondisjunction events, resulting in loss of the full-length Chr7 homolog and loss of the fusion Chr5,6 (114). (D) aCGH analysis indicates that strain WO-1 does not contain obvious whole-chromosome or segmental aneuploidies, although some isolates of WO-1 do carry a trisomy of Chr1 (149). (E) In contrast, WO-2 carries a total of four segmental aneuploidies, including segmental trisomy of Chr4 (the result of duplication of the Chr7,4 fusion chromosome) and segmental monosomy of Chr5, -6, and -7 (after loss of the full-length Chr7 homolog and loss of the Chr5,6 fusion chromosome). Note that the transition points for the segmental aneuploidies all occur at or very close to the MRS (pink ovals). aCGH analyses of WO-1 and WO-2 have not been published previously.

Fig. 5.

Comparative genome hybridization analysis of several well-characterized laboratory strains. (A) CAI4 isolate F3 is trisomic for Chr1 and -2, and other CAI4 isolates are trisomic only for Chr2 (172). (B) BWP17 has a segmental monosomy of Chr5 distal to the HIS1 locus. (C) aCGH of strain 3153A does not detect chromosome alterations for ChrR, -5, and -6 (157); (D) aCGH of SGY-243 detects only trisomy of ChrR (64) and Chr2, but neither Chr1 (28, 111) nor Chr4, -5, -6, or -7 (157) appear to be aneuploid in our collection; and (E) aCGH of Sou⁺ strain Sor5 detects two segmental aneuploidies: segmental monosomy of Chr5R as well as a segmental trisomy of Chr4R. The sorbose utilization genes SOU1 and SOU2 are located within this trisomic region and likely contribute to the ability of the strain to grow on sorbose. (F to H) Auxotrophic strains SN87, SN95, and SN152 (137), derived from RM1000#2, are disomic. Data in panels C to H have not been published previously.