Molecular genetic analyses of mating pheromones reveal intervariety mating or hybridization in Cryptococcus neoformans

Abstract

The sexual mating of the pathogenic yeast Cryptococcus neoformans is important for pathogenesis studies because the fungal virulence is linked to the alpha mating type (MAT(alpha)). We characterized C. neoformans mating pheromones (MF(alpha) 1 and MFa1) from 122 strains to understand intervariety hybridization or mating and intervariety virulence. MF(alpha) 1 in three C. neoformans varieties showed (a) specific nucleotide polymorphisms, (b) different copy numbers and chromosomal localizations, and (c) unique deduced amino acids in two geographic populations of C. neoformans var. gattii. MF(alpha) 1 of different varieties cross-hybridized in Southern hybridizations. Their phylogenetic analyses showed purifying selection (neutral evolution). These observations suggested that MAT(alpha) strains from any of the three C. neoformans varieties could mate or hybridize in nature with MATa strains of C. neoformans var. neoformans. A few serotype A/D diploid strains provided evidence for mating or hybridization, while a majority of A/D strains tested positive for haploid MF(alpha) 1 identical to that of C. neoformans var. grubii. MF(alpha) 1 sequence and copy numbers in diploids were identical to those of C. neoformans var. grubii, while their MFa1 sequences were identical to those of C. neoformans var. neoformans; thus, these strains were hybrids. The mice survival curves and histological lesions revealed A/D diploids to be highly pathogenic, with pathogenicity levels similar to that of the C. neoformans var. grubii type strain and unlike the low pathogenicity levels of C. neoformans var. neoformans strains. In contrast to MF(alpha) 1 in three varieties, MFa1 amplicons and hybridization signals could be obtained only from two C. neoformans var. neoformans reference strains and eight A/D diploids. This suggested that a yet undiscovered MFa pheromone(s) in C. neoformans var. gattii and C. neoformans var. grubii is unrelated to, highly divergent from, or rarer than that in C. neoformans var. neoformans. These observations could form the basis for future studies on the role of intervariety mating in C. neoformans biology and virulence.

FIG. 1.

Characterization of MFα1 nucleotide sequences. The consensus sequences representing three different varieties were obtained from the alignment of MFα1 sequences of all test isolates by using a GCG software package. C. neoformans var. gattii sequences of the isolates from California (CA) and Australia (AU) revealed population-specific substitutions.

FIG. 2.

Copy numbers of MFα1 in type strains of three C. neoformans varieties. Southern blots of genomic DNA digested with HaeII and probed with ³²P-labeled, 101-bp PCR fragments of MFα1 from C. neoformans var. grubii (H99) are shown. Lambda DNA digested with HindIII was used as a size marker. NIH 12 (C. neoformans var. neoformans, serotype D, MATα) showed three bands and H99 (C. neoformans var. grubii, serotype A, MATα) revealed four bands, while NIH 444 (C. neoformans var. gattii, serotype B, MATα) yielded two bands. The MATa strains (NIH 430, NIH 433, and NIH 191) showed no hybridization signal, confirming the specificity of the MFα1 probe.

FIG. 3.

Neighbor-joining phylogenetic tree of MFα1. C. neoformans var. grubii, C. neoformans var. gattii, and C. neoformans var. neoformans formed three separate clades, which were strongly supported by high bootstrap values. MFα1A/D segregated with C. neoformans var. grubii. Branch length comparisons indicated that C. neoformans var. grubii was greatly separated from the other two varieties. Both C. neoformans var. grubii and C. neoformans var. neoformans were monophyletic. Interestingly, C. neoformans var. gattii showed three sister groups, with California isolates in one cluster and the Australian isolates in the other two clusters. However, low bootstrap values precluded any definitive interpretation of this subgrouping in C. neoformans var. gattii.

FIG. 4.

Copy numbers of MFa1 in type strains of three C. neoformans varieties. Southern blots with ³²P-labeled, 117-bp PCR-amplified MFa1 fragments from C. neoformans var. neoformans (NIH 430). Genomic DNA was digested with BamHI; lambda DNA digested with HindIII was used as a size marker. MFa1 hybridization of restriction-digested genomic DNA showed three distinct bands in NIH 430 and NIH 433. All MATα strains were negative, thus confirming the specificity of the probe. Interestingly, NIH 191 (MATa, serotype C) was also negative, suggesting a widely divergent MATa in C. neoformans var. gattii.

FIG. 5.

A/D diploid MFα1 and MFa1 copy numbers. Genomic DNA was digested with HaeII or BamHI and hybridized with labeled MFα1 or MFa1 probes as described for Fig. 2 and 4. (A) Three distinct MFα1 bands in strain NIH 12 (MATα, serotype D) and four MFα1 bands in C. neoformans var. grubii type strain H99 (MATα, serotype A) as well as in all of the eight A/D isolates are shown. (B) Three MFa1 bands for the two C. neoformans var. neoformans strains NIH 430 and NIH 433 (MATa, serotype D) are shown; A/D isolates showed two bands.

FIG. 6.

Morphometry of SEM images was used to compare the cell sizes of A/D isolates with those of C. neoformans var. grubii MATα type strain H99 or C. neoformans var. neoformans MATa type strain NIH 430 (details in Materials and Methods). The box graph illustrates average cell sizes, with median values marked by horizontal lines; positive and negative standard deviations are also shown along with outlier values for 50 cell sizes. Cells of all diploid strains were significantly larger than those of the two control strains (Student t test; P < 0.001).

FIG. 7.

Survival curves of mice infected with C. neoformans strains. Groups of five mice received 10⁶ C. neoformans cells intravenously, and the course of infection was followed over 30 days. C. neoformans H99 (C. neoformans var. neoformans, MATα) was the most lethal strain, while the C. neoformans var. neoformans isogenic mating pair (JEC20 and JEC21) caused no mortality. Most A/D strains caused infections similar to that caused by H99.

FIG.8.

Brain sections from infected mice. In groups of two or three, mice were infected with 10⁶ C. neoformans cells intravenously and euthanized on day 7, and the brain sections were stained with Mayers mucicarmine. Few, scattered, and small foci with yeasts of control strain JEC20 are seen in the grey and white matter of cortex and diencephalons (A; ×20 magnification). A close-up of small thalamic cysts shows expansion in closely apposed perivascular spaces by yeasts (a; ×200). A section from another control strain (H99) shows numerous large cysts scattered in many parts of the brain (B; ×20), and higher magnification of a large thalamic perivascular cyst shows it to be full of yeasts, with scant inflammatory reaction (b; ×200). The cerebrum, with thalamus and lateral ventricle, of a mouse inoculated with a representative diploid 190C strain shows scattered cysts containing numerous yeasts (C; ×20). A close-up of cyst-like, confluent perivascular spaces filled with numerous yeasts is shown (c; ×200).