Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Abstract

Background: Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results: Initially, CGH was used to compare the approximately 100 kilobase MATa and MATalpha mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MATa) and the clinical isolate NIH12 (MATalpha) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion: The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.

Figure 1

Comparisons of Log2 ratios and sequence identity for the MATa and MATα loci of serotype A and D strains. Each data point represents the average Log2 ratio and sequence similarity of eight probes from a set of 400 base pair windows within 20 genes at the MAT locus. (a) Comparison of the MAT loci of the serotype D strains JEC20 (MATa) and JEC21 (MATα). (b) Comparison of the MAT loci of the serotype A strains 125.91 (MATa) and H99 (MATα). Note that the MATa region of JEC20 originated in strain NIH433 and genomic DNA of NIH433 was used for the hybridization experiment.

Figure 2

Genome hybridization to compare the progenitor strains NIH12 and NIH433 to the reference strain JEC21. Regions with higher variability in Log2 ratios in the test genomes are more divergent from the JEC21 sequence; regions with Log2 ratios close to zero have greater similarity (Additional data file 1). A reciprocal pattern of similar and divergent segments is found upon hybridization of genomes of NIH12 and NIH433 to the JEC21 array. The scale of chromosome coordinates for the JEC21 genome is indicated at the top of the figure, and gaps in the chromosomes represent putative centromeric regions [46]. The borders of segments are probable sites of recombination events that occurred during the mating of NIH12 and NIH433, and the subsequent backcrossing to obtain JEC21 (Additional data file 2) [59].

Figure 3

Sequence divergence and putative recombination sites at the MAT locus in serotype D strains. The chromosomal coordinates are shown at the top and the annotated genes in the region are indicated as boxes. Putative recombination sites are marked with arrows. Specific genes that are known to have high sequence similarity between the MATa and MATα alleles [15], and that exhibit a corresponding Log2 ratio close to zero are labelled with letters: a, RPO41 (XM_570485.1; The Institute for Genomic Research [TIGR] locus tag CND05820); b, BSP2 (XM_570482.1; TIGR locus tag CND05830); c, LPD1 (XM_570114.1; TIGR locus tag CND05840); d, CID1 (XM_570548.1; TIGR locus tag CND05850); and e, GEF1 (XM_570546.1; TIGR locus tag CND05860). The gap in the hybridization signal for NIH12 and NIH433 centered on position 1,610,000 resulted from the presence of repeated sequences in this region that reduced probe density [15].

Figure 4

Variation in chromosomes 1, 5, and 13 for four serotype A strains. The DNA from the strains was hybridized to the array from the genome of strain H99, and gaps in the chromosomes represent the positions of repetitive sequences that represent putative centromeres or repeated elements in the MAT locus. The spikes in the Log2 ratios (for instance, for chromosome 1 of CBS7779) represent individual probes with high Log2 ratios (3 to 4); the sequences of these probes are present in single copy in the H99 genome but may be part of repetitive sequences in the other strains. For all of the chromosomes in all four strains, the hybridization data are shown in Additional data file 10, the Log2 ratios are listed in Table 1 and the regions of difference are listed in Additional data file 3.

Figure 5

Hybridization analysis of three AD hybrid strains. The chromosome (chr) numbers listed at the bottom of each panel follow those of the reference genomes on the tiling arrays; for example, the chromosome numbers from JEC21 are used for the hybridization of DNA from each strain to the JEC21 array. Note that the JEC21 and H99 genomes are largely co-linear, but some of the homologous chromosomes have been assigned different numbers in the current genome assemblies [61]. Most of the chromosomes of the AD hybrid strains are represented by copies from both A and D genomes. However, the hybridization signals indicate that chromosome 1 is only represented by sequences from a serotype A genome in all three strains. Similarly, chromosomes 5, 6, and 7 (and chromosome 14 in KW5) are represented by sequences from only one of the serotypes (either A or D). The average Log2 ratios and standard deviations for all of the chromosomes are listed in Additional data file 6, and PCR-RFLP confirmation for selected chromosomes is shown in Figure 6.

Figure 6

PCR-RFLP confirmation of the presence of serotype-specific chromosomes in three AD hybrid strains. Agarose gels are shown in which lane 1 for each contains size markers (1 kilobase [kb] ladder); lanes 2, 4, 6, 8, and 10 contain undigested PCR fragments; and the remaining lanes contain the same fragments after restriction enzyme digestion. (a) Digestion of PCR fragments (primers CNA01230 F/R) from chromosome (chr) 1 with AvaI (left panel) or StuI (right panel). (b) Digestion of PCR fragments (primers CNE04380 F/R) from chromosome 5 with TaqI (left panel) or HindIII (right panel). (c) Digestion of a PCR fragment (primers CNB01970 F/R) from chromosome 2 with NdeI (left panel) and a fragment (primers acidphos F/R) from chromosome 3 with TaqI (right panel).