A unique chromosomal rearrangement in the Cryptococcus neoformans var. grubii type strain enhances key phenotypes associated with virulence

Abstract

The accumulation of genomic structural variation between closely related populations over time can lead to reproductive isolation and speciation. The fungal pathogen Cryptococcus is thought to have recently diversified, forming a species complex containing members with distinct morphologies, distributions, and pathologies of infection. We have investigated structural changes in genomic architecture such as inversions and translocations that distinguish the most pathogenic variety, Cryptococcus neoformans var. grubii, from the less clinically prevalent Cryptococcus neoformans var. neoformans and Cryptococcus gattii. Synteny analysis between the genomes of the three Cryptococcus species/varieties (strains H99, JEC21, and R265) reveals that C. neoformans var. grubii possesses surprisingly few unique genomic rearrangements. All but one are relatively small and are shared by all molecular subtypes of C. neoformans var. grubii. In contrast, the large translocation peculiar to the C. neoformans var. grubii type strain is found in all tested subcultures from multiple laboratories, suggesting that it has possessed this rearrangement since its isolation from a human clinical sample. Furthermore, we find that the translocation directly disrupts two genes. The first of these encodes a novel protein involved in metabolism of glucose at human body temperature and affects intracellular levels of trehalose. The second encodes a homeodomain-containing transcription factor that modulates melanin production. Both mutations would be predicted to increase pathogenicity; however, when recreated in an alternate genetic background, these mutations do not affect virulence in animal models. The type strain of C. neoformans var. grubii in which the majority of molecular studies have been performed is therefore atypical for carbon metabolism and key virulence attributes.

Importance: The fungal pathogen Cryptococcus is a major cause of mortality among the immunocompromised population, primarily in AIDS patients of sub-Saharan Africa. Most research into the particular variety of Cryptococcus responsible for the vast majority of infections, Cryptococcus neoformans var. grubii, is performed using the type strain isolated in 1978 from a Hodgkin's disease patient from North Carolina. We have determined that this particular isolate contains a chromosomal translocation that directly interrupts two genes, which all descendants of this strain from various research laboratories appear to possess. Disruption of these two genes affects multiple virulence factors of Cryptococcus, particularly the ability to grow at human body temperature, which could have wide-ranging implications for molecular genetic studies and virulence assays using this important strain.

FIG 1

Inversions unique to the C. neoformans var. grubii genome. Green regions are syntenic with the C. neoformans var. neoformans and C. gattii genomes, while red regions are inverted and yellow indicates the duplication. Genes are represented by black arrows indicating direction of transcription, while white arrowheads represent transposable or repeated elements near inversion breakpoints. Images are not to scale. CoA, coenzyme A.

FIG 2

Identification and reconstruction of the H99 translocation. (A) Diagnostic PCR assay spanning the translocation boundary designed to produce an amplicon only when the translocation is present. Extended testing establishes that the translocation is present in all subcultures of H99 tested but absent from all other strains. (B) Alignment of the translocation breakpoints from H99, JEC21, R265, and VNI isolate 125.91. Note the presence of a microhomology, AGC, at the breakpoint, along with a single-base-pair insertion, A, before the microhomology on H99 chromosome 11. In silico reversal of the translocation and removal of the base pair insertion yields two complete ORFs without frameshift or nonsense mutations.

FIG 3

Reverse genetics in strain Bt63 and phenotypic characterization reveal roles for the two genes disrupted by the translocation observed in strain H99. (A) The two genes disrupted by the translocation, designated YHP2 and TGR1, were deleted via homologous recombination in the VNB isolate Bt63. Tenfold serial dilutions of each strain were spotted onto YNB plates supplemented with the indicated carbon source, stressor, or osmotic stabilizer. The complemented strain Bt63 tgr1Δ TGR1 displayed the wild-type Bt63 phenotype for all assays where it is not shown. (B) Polysaccharide capsule production visualized as a clear halo around the cell in an India ink stain. Bar = 10 µm. (C) Melanization of strains on l-DOPA-containing medium. Melanized Cryptococcus cells turn brown-black.

FIG 4

Expression profiling of TGR1 and genes involved in high-temperature growth and stress responses. Indicated strains were grown on YNB plus glucose or galactose plates for 2 days. (A) Gene expression analysis of TGR1 in Bt63 at 30°C or 37°C. (B) Expression analysis of trehalose biosynthesis genes and the two hexokinases at 37°C. (C) Gene expression quantification of selected genes implicated in high-temperature growth or cellular stress responses at 37°C. Bars represent means ± standard errors of gene expression relative to that of β-actin (ACT1) from three biological replicates and three technical replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Student’s two-tailed t test, two sample, equal variance).

FIG 5

Metabolomics analysis of Bt63 and the tgr1Δ mutant strain. (A) 1D proton NMR spectra of wild-type (top, blue) and tgr1Δ mutant (bottom, red) strains. Spectra of representative samples for each strain are shown. (B) Principal component analysis scores plot for the wild-type and tgr1Δ mutant strains. The distance between the points is an indicator of similarity between the samples. (C) Bivariate loadings line plot. The line plot displays on the loadings coefficient axis the correlation coefficients that relate individual integral regions of the NMR spectrum to the PC2 axis of the scores plot. Individual peaks correspond to peaks in the 1D NMR spectra; here, peaks that are positive indicate metabolites that are increased in the wild-type Bt63 while negative peaks are increased in the tgr1Δ mutant. The overlaid heat map relates the correlation of the peak to the PC2 axis of the scores plot when using Pareto scaling instead of center scaling.

FIG 6

Virulence of translocation gene mutants in the nematode and murine inhalation models of cryptococcosis. (A) Survival times of N2 Bristol nematodes cultured on Bt63, tgr1Δ, tgr1Δ TGR1, yhp2Δ, yhp2Δ YHP2, and tgr1Δ yhp2Δ strains. (B) Virulence of translocation mutant strains tested in vivo using a mouse model system. No nematodes or mice infected with mutant strains were significantly different from those infected with wild-type Bt63.