Genetic Diversity and Genomic Plasticity of Cryptococcus neoformans AD Hybrid Strains

Abstract

Natural hybridization between two strains, varieties, or species is a common phenomenon in both plants and animals. Although hybridization may skew established gene pools, it generates population diversity efficiently and sometimes results in the emergence of newly adapted genotypes. Cryptococcus neoformans, which causes the most frequent opportunistic fungal infection in immunocompromised hosts, has three serotypes: A, D, and AD. Serotype-specific multilocus sequence typing and serotype-specific comparative genome hybridization were applied to investigate the genetic variability and genomic organization of C. neoformans serotype AD isolates. We confirm that C. neoformans serotype AD isolates are hybrids of serotype A and D strains. Compared with haploid strains, most AD hybrid isolates exhibit unique multilocus sequence typing genotypes, suggesting that multiple independent hybridization events punctuated the origin and evolutionary trajectory of AD hybrids. The MATa alleles from both haploid and AD hybrid isolates group closely to form a cluster or subcluster in both the serotype A and D populations. The rare and unique distribution of MATa alleles may restrict sexual reproduction between isolates of opposite mating types. The genetic diversity of the serotype D population, including haploid strains and serotype D genomes of the AD hybrid, is significantly greater than that of serotype A, and there are signatures of recombination within the serotype D population. Given that MATa isolates are relatively rare, both opposite-sex and same-sex mating may contribute to genetic recombination of serotype D in nature. Extensive chromosome loss was observed in AD hybrid isolates, which results in loss of heterozygosity in the otherwise-heterozygous AD hybrid genome. Most AD hybrid isolates exhibit hybrid vigor and are resistant to the antifungal drug FK506. In addition, the C. neoformans AD hybrid genome is highly dynamic, with continuous chromosome loss, which may be a facile route for pathogen evolution through which genotypic and phenotypic variation is generated.

Figure 1

C. neoformans AD hybrid isolates have similar DNA content to diploid strains. DNA content was measured by FACS analysis. The haploid strain H99 and the diploid strain XL143 were used as 1N and 2N controls, respectively. The C. neoformans AD hybrid isolates AD7-97, AD2-71, and IUM92-6198 have similar DNA content as diploid control strain XL143 and serotype DD diploid strain AD8-62.

Figure 2

Phylogenetic organization of C. neoformans serotype A (left) and serotype D (right). This analysis was based on the concatenation of 5 MLST markers: IGS, URE1, GPD1, LAC1, and MPD1. The five loci were specifically amplified and sequenced from the serotype A and D genomes of the AD hybrid isolate by the use of serotype-specific primers. For comparison with haploid strains, 32 serotype D haploid strains, 1 serotype DD diploid strains, and 7 serotype A strains (*) of VNI, VNII, and VNB lineages from a previous study (Litvintseva et al. 2006) were included in the phylogenetic analysis. The concatenated DNA sequences were aligned by the use of ClustalW and MUSCLE. The phylogenetic organization was constructed with Mega 3.1 via the neighbor-joining method. The MATa strains are shown in red. The serotype and mating type of each isolate are indicated in brackets.

Figure 3

Extensive chromosome loss in C. neoformans AD hybrid isolates. Genomic DNA of the AD hybrid isolate and the control (equal amount of genomic DNA of H99 and JEC21) were competitively hybridized to 70-mer oligonucleotide probes designed from the JEC21 and H99 genomes. The serotype-specific probes on the array slide were determined on the basis of bioinformatic analysis of the probe sequences against the H99 and JEC21 genomic sequences (see Materials and methods section). The hybridization signal of serotype-specific probes was analyzed by the use of GenePix and GeneSpring software to produce the log2 ratio of R635/R532 (AD hybrid/control) value and plotted in Microsoft Excel. Each block of different color represents a chromosome. For the serotype D genome, from left to right the 14 blocks match chromosomes 1 to 14. Serotype A chromosome numbers are redefined according to the corresponding serotype D chromosome (for the corresponding serotype A and D chromosomes, see Figure S1). Each triangle represents a serotype-specific probe. The line in each block (chromosome) represents the average log2 ratio of genomic hybridization. Average log2 ratios lower and higher than 0.5 reflect chromosome loss and duplication, respectively. For example, strain IUM92-6198 lost serotype D Chr 9, and serotype A Chr 3, Chr5, Chr 8, and Chr 11. Strain AD2-71 has partial loss of serotype A Chr 1, Chr 2, and Chr 3.

Figure 4

Quantitative analysis of gene copy number of AD hybrid isolates. Heterozygous loci of AD hybrid isolates have two copies of a gene, one from the serotype A parent and the other one from the serotype D parent. The primers used for quantitative analysis of the gene copy number of the AD hybrid isolates are located in regions conserved between serotype A and D and can amplify both serotype A and D loci. The primers target three loci on Chr 1, Chr 4, and Chr 9. On the basis of CGH, strain IUM92-6198 has lost serotype D Chr 9, and strains KW5 and CDC228 have lost serotype D Chr 1; however, these strains have an identical copy number of genes on Chr 1, Chr 4, and Chr 9. This finding indicates chromosome duplication of the remaining chromosomes after chromosome loss from one parental serotype in the AD hybrid.

Figure 5

The serotype AD population exhibits genotypic plasticity (2N to 2N-1 to 2N). Two populations are derived from KW5: one with and one without serotype A Chr 14. The serotype A URE1 gene, located on serotype A Chr 14, was amplified from three clones (C2, C3, and C7) from KW5 but not the other 5 clones. All eight clones have the same serotype/mating type, αADa, based on serotype/mating type determination via the use of PCR assays of the STE20 gene. CGH analysis demonstrates that clone C5 has lost serotype A Chr 14, which is extant in the C7 colony.

Figure 6

The progeny of self-fertile strain CDC228 (aADα) have highly diverse genome organizations compared with the parental strain. Progeny P5, which is sensitive to the antifungal drug FK506 (Figure 7), has lost serotype D Chr 3 and Chr 12, and serotype A Chr 5, Chr 8 (partial), Chr 10 (partial), and Chr 13 compared with the parental strain CDC228, which only lost the serotype D copy of Chr 1 based on CGH. The P5 progeny has a distinct genomic profile based on CHEF analysis. It lacks some chromosomes (arrows) compared with CDC228.

Figure 7

AD hybrid isolates exhibit phenotypic plasticity. Most AD hybrid isolates are resistant to the antifungal drug FK506 (left) and the progeny of the self-fertile strain CDC228 (aADα), which is resistant the antifungal drug FK506, have altered FK506 resistance (right). After growing for 48 hr on YPD + FK506 (1 µg/ml) medium at 37°, five AD hybrid isolates (ZG290, NC34-21, MMRL1365, MMRL774, and MMRL752) exhibited resistance compared with the H99 and JEC21 haploid strains (left). After growing for 48 hr on YPD + FK506 (1 µg/ml) medium at 37°C, progeny P5 was sensitive to FK506 (right).