Isolates of Cryptococcus neoformans from infected animals reveal genetic exchange in unisexual, alpha mating type populations

Abstract

Sexual reproduction and genetic exchange are important for the evolution of fungal pathogens and for producing potentially infective spores. Studies to determine whether sex occurs in the pathogenic yeast Cryptococcus neoformans var. grubii have produced enigmatic results, however: basidiospores are the most likely infective propagules, and clinical isolates are fertile and genetically diverse, consistent with a sexual species, but almost all populations examined consist of a single mating type and have little evidence for genetic recombination. The choice of population is critical when looking for recombination, particularly when significant asexual propagation is likely and when latency may complicate assessing the origin of an isolate. We therefore selected isolates from infected animals living in the region of Sydney, Australia, with the assumption that the relatively short life spans and limited travels of the animal hosts would provide a very defined population. All isolates were mating type alpha and were of molecular genotype VNI or VNII. A lack of linkage disequilibrium among loci suggested that genetic exchange occurred within both genotype groups. Four diploid VNII isolates that produced filaments and basidium-like structures when cultured in proximity to an a mating type strain were found. Recent studies suggest that compatible alpha-alpha unions can occur in C. neoformans var. neoformans populations and in populations of the sibling species Cryptococcus gattii. As a mating type strains of C. neoformans var. grubii have never been found in Australia, or in the VNII molecular type globally, the potential for alpha-alpha unions is evidence that alpha-alpha unisexual mating maintains sexual recombination and diversity in this pathogen and may produce infectious propagules.

FIG. 1.

UPGMA phylogram derived from AFLP data. The phylogram shows the relatedness of isolates included in the population, mating abilities, and ploidies (1N, haploid; 2N, diploid). To assess the mating ability, filaments, basidia, and basidiospores were scored from + to +++++ according to whether they were few to very profuse (see Materials and Methods for a complete description of scoring). − indicates the absence of the structure.

FIG. 2.

Distributions of I_A and T_L values. Histograms show the ranges of I_A and T_L values obtained for 1,000 randomizations of the observed data sets for the VNI and VNII populations. Observed I_A and T_L values for each population lie within the randomized data sets.

FIG. 3.

Analysis of the diploid C. neoformans strains. Diploid (2N) isolates 571-154 and 571-216 are shown alongside haploid (1N) strains JEC21 (C. neoformans var. neoformans; α mating type) and 571-168 (C. neoformans var. grubii [this study]). (A) Filament formation in the confrontation assay with the a mating type C. neoformans serotype D strain JEC20. Diploids produced extensive filaments, which were not produced by JEC20 or by any of the haploid isolates. (B) FACS analysis indicated that self-filamenting strains have diploid genomes. 4N, quadriploid. (C) DIC microscopy images. Diploid cells had the morphology typical of C. neoformans encapsulated yeast cells but were slightly larger than haploids. (D) Fluorescence microscopy images of the same field shown in panel C. Diploid cells had a single nucleus.

FIG. 4.

Mating structures produced by diploids. Diploid isolates produced swollen hyphal tips resembling basidia but no spores when grown in confrontation with the a mating type strain. (A) Basidium-like structures produced by diploid isolate 571-154 grown in confrontation with JEC20. (B) The same isolate produced basidia and extensive spores following coculture and mating with JEC20.

FIG. 5.

Phylogenetic analysis of representative isolates compared with global VNI and VNII strains. The maximum-parsimony tree was derived from the sequences of six unlinked genetic loci. Isolates included in this study are shown in boldface; D indicates diploids. Australian VNII veterinary isolates formed a clade that was distinct from other VNII isolates.