Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts

Abstract

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

FIG 1

Alignments of colinear and rearranged chromosomes. (A) Alignments of the sequence of chromosome 4 from the two C. gattii strains, WM276 and R265. (B) Alignment of chromosome 4 from C. gattii strain WM276 with the corresponding sequences from the C. neoformans strain B3501A, which are present on chromosomes 4, 9, and 10. Aligned chromosomes are indicated by green lines. The blue bar above each chromosome pair shows the percent sequence identity (panel A, ~92.4%; panel B, ~85%; overall percentage indicated by the red dashed line). The blue and pink segments between the chromosomes indicate whether the alignments are direct or inverted, respectively (blocks are bounded by a darker color and filled with a lighter color).

FIG 2

Multiortholog phylogeny of Cryptococcus strains and other basidiomycete fungi. Branch lengths of the indicated phylogenetic trees represent the number of nucleotide substitutions per site calibrated in million year divergence times (shown above the branches), and the numbers in brackets are values for bootstrap support of the branch. (A) A calibrated phylogenetic tree based on 5,171 single-copy orthologs conserved between the five indicated Cryptococcus strains was generated using the age estimate of the most recent common ancestor of the C. gattii-C. neoformans var. grubii lineages. Genome alignment results and the extent of divergence (12.4 myr) between the VGI and VGII C. gattii strains advocate for the existence of speciation between these molecular types. (B) A phylogenetic tree based on 1,519 single-copy orthologs conserved among the 10 indicated fungal species was calibrated based on a recent estimate of ~500 myr of divergence between ascomycetous and basidiomycetous fungi. According to this calibration, the two C. gattii molecular types would have diverged about 11 myr ago. It appears that the cryptococci, representatives of the class Tremellomycetes, diverged about 291 myr from the common ancestors of Phanerochaete chrysosporium and Coprinus cinereus.

FIG 3

Virulence of Vancouver Island isolates of C. gattii. Ten A/JCr female mice were inoculated intranasally with 5 × 10⁴ cells of each of the strains indicated and monitored for illness over 2 months. These assays were performed twice for all of the strains, with similar results. The analyses of virulence for strains WM276 (VGI), R265 (VGIIa), and R272 (VGIIb) (shown here only for comparison) were performed in the same experiment and previously published (40). The differences in virulence for WM276 versus those in the VGI strains R794 (P < 0.0001) and KB3864 (P < 0.0001) were statistically significant, and the mice infected with WM276 reached the endpoint of the experiment at day 36. The virulence of VGIIb strain RB28 was attenuated compared with that of VGIIb strain R272 (endpoint at day 36) and relative to that of the more virulent VGIIa strain R265 (endpoint at day 23) (40). The lower virulence of R272 relative to that of R265 has also been described in other studies (24, 40, 75, 76)

FIG 4

Comparative hybridization of fluconazole-resistant VGI isolates of C. gattii strains from the VGI and VGII molecular types. DNA from all resistant strains and their cognate parental strains was differentially labeled with fluorescent dyes prior to competitive hybridization to the VGI reference genome (WM276) or the VGII reference genome (R265) array. Log₂ ratios for assessing relative hybridization were averaged in windows of 400 bp. (A) VGI strains R1412F and R1413F (supported by FACS analysis) (see Text S1, p. 31 in the supplemental material). (B) VGII strains R1347F and R1402F (supported by FACS analysis) (see Text S1, p. 32 and 33 in the supplemental material). CGH plots for R1401F and R1346F can be found in Text S1, p. 30 in the supplemental material (see FACS analysis in Text S1, p. 32 and 33 in the supplemental material).