Unisexual and Heterosexual Meiotic Reproduction Generate Aneuploidy and Phenotypic Diversity De Novo in the Yeast Cryptococcus neoformans

Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down's syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast Cryptococcus neoformans, which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. C. neoformans has a defined a-α opposite sexual cycle; however, >99% of isolates are of the α mating type. Interestingly, α cells can undergo α-α unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that α-α unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from a-α opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity de novo. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments

Amoeba predation of <i>Cryptococcus</i>:A quantitative and population genomic evaluation of the accidental pathogen hypothesis

The “Amoeboid Predator-Fungal Animal Virulence Hypothesis” posits that interactions with environmental phagocytes shape the evolution of virulence traits in fungal pathogens. In this hypothesis, selection to avoid predation by amoeba inadvertently selects for traits that contribute to fungal escape from phagocytic immune cells. Here, we investigate this hypothesis in the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans. Applying quantitative trait locus (QTL) mapping and comparative genomics, we discovered a cross-species QTL region that is responsible for variation in resistance to amoeba predation. In C. neoformans, this same QTL was found to have pleiotropic effects on melanization, an established virulence factor. Through fine mapping and population genomic comparisons, we identified the gene encoding the transcription factor Bzp4 that underlies this pleiotropic QTL and we show that decreased expression of this gene reduces melanization and increases susceptibility to amoeba predation. Despite the joint effects of BZP4 on amoeba resistance and melanin production, we find no relationship between BZP4 genotype and escape from macrophages or virulence in murine models of disease. Our findings provide new perspectives on how microbial ecology shapes the genetic architecture of fungal virulence, and suggests the need for more nuanced models for the evolution of pathogenesis that account for the complexities of both microbe-microbe and microbe-host interactions

Discovery of a Modified Tetrapolar Sexual Cycle in Cryptococcus amylolentus and the Evolution of MAT in the Cryptococcus Species Complex

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The human fungal pathogenic and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system (a, α) in which the MAT locus is unusually large (>100 kb) and encodes >20 genes including homeodomain (HD) and pheromone/receptor (P/R) genes. To understand how this unique bipolar mating system evolved, we investigated MAT in the closely related species Tsuchiyaea wingfieldii and Cryptococcus amylolentus and discovered two physically unlinked loci encoding the HD and P/R genes. Interestingly, the HD (B) locus sex-specific region is restricted (∼2 kb) and encodes two linked and divergently oriented homeodomain genes in contrast to the solo HD genes (SXI1α, SXI2a) of C. neoformans and Cryptococcus gattii. The P/R (A) locus contains the pheromone and pheromone receptor genes but has expanded considerably compared to other outgroup species (Cryptococcus heveanensis) and is linked to many of the genes also found in the MAT locus of the pathogenic Cryptococcus species. Our discovery of a heterothallic sexual cycle for C. amylolentus allowed us to establish the biological roles of the sex-determining regions. Matings between two strains of opposite mating-types (A1B1×A2B2) produced dikaryotic hyphae with fused clamp connections, basidia, and basidiospores. Genotyping progeny using markers linked and unlinked to MAT revealed that meiosis and uniparental mitochondrial inheritance occur during the sexual cycle of C. amylolentus. The sexual cycle is tetrapolar and produces fertile progeny of four mating-types (A1B1, A1B2, A2B1, and A2B2), but a high proportion of progeny are infertile, and fertility is biased towards one parental mating-type (A1B1). Our studies reveal insights into the plasticity and transitions in both mechanisms of sex determination (bipolar versus tetrapolar) and sexual reproduction (outcrossing versus inbreeding) with implications for similar evolutionary transitions and processes in fungi, plants, and animals

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence

Unisexual and Heterosexual Meiotic Reproduction Generate Aneuploidy and Phenotypic Diversity <i>De Novo</i> in the Yeast <i>Cryptococcus neoformans</i>

<div><p>Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down's syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast <i>Cryptococcus neoformans</i>, which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. <i>C. neoformans</i> has a defined <b>a</b>-α opposite sexual cycle; however, >99% of isolates are of the α mating type. Interestingly, α cells can undergo α-α unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that α-α unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from <b>a</b>-α opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity <i>de novo</i>. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling <i>de novo</i> genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments.</p></div

Aneuploidy causes the observed phenotypic changes.

<p>Strain MN77, which contains an extra copy of Chr 10 and has increased melanin production, was grown on YPD at 37°C for 3 d to promote aneuploidy loss. The genotypes and phenotypes of 22 resulting mitotic progeny were analyzed. (A) Melanin production of 22 mitotic progeny on NS media at 30°C for 2 d. (B) Multiplex PCR of 22 progeny detected an extra Chr 10 in all of the strains with the increased melanin phenotype and loss of the extra copy of Chr 10 in all progeny with a wild-type phenotype. (C) Phenotypic analysis of 22 mitotic progeny at high-temperature growth (37°C for 2 d) and with drug treatment (FLC and FK506). XL280 (X) and MN77 (77) are the WT and aneuploid controls, respectively.</p

Aneuploidy is generated at a high rate by unisexual reproduction.

<p>(A) CGH array data for four progeny with novel phenotypes. Progeny strains MN35, MN55, MN77, and MN89 contain an extra copy of Chr 13, Chr 9, Chr 10, and Chr 10, respectively. The colors indicate gene dosage as follows: gray, no significant change; red, more abundant; green, less abundant. (B) NGS is consistent with the CGH data in that 2× coverage was observed for each aneuploid chromosome and no others. MN55 (yellow) has an extra copy of Chr 9, which corresponds to the left arm of Chr 8 and the right arm of Chr 12 of strain JEC21 (as shown above). MN89 (blue) has an extra copy of Chr 10. (C) Multiplex PCR was conducted to detect aneuploidy. Multiplex PCR reactions used the primer sets listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001653#pbio.1001653.s018" target="_blank">Table S3</a>. Amplicons (i.e., chromosomes or chromosome regions) that differed in abundance between samples were identified in this assay and quantified by scanning (arrowheads). Chromosome numbers are indicated on the left.</p

Unisexual reproduction generates phenotypic diversity.

<p>(A) Progeny produced by α-α unisexual reproduction of strain XL280 in solo cultures were grown in 10-fold serial dilution assays and under the following conditions: YPD at 30°C for 2 d; YPD at 37°C for 2 d; YPD plus 1 µg/mL FK506 at 30°C for 2 d; YPD plus 8 µg/mL fluconazole (FLC) at 30°C for 6 d; NS (melanin-inducing media) at 30°C for 3 d; and V8 pH = 7 (mating-inducing media) at room temperature for 11 d. (B) Diploid progeny were generated infrequently following unisexual reproduction. Flow cytometry profiles of cells stained with the fluorescent dye propidium iodide. JEC21 (1n, haploid control); XL143 (2n, diploid control); XL280 (1n); MN27 (2n, the only diploid strain identified among the 90 XL280 α-α unisexual reproduction progeny (1.1%). All other XL280 α-α unisexual progeny were haploid (or aneuploid) (e.g., MN55). Nuclear DNA content is indicated by 1n (haploid) and 2n (diploid). The <i>x</i>-axis indicates fluorescence intensity reflecting DNA content, and the <i>y</i>-axis indicates cell counts. (C) Three SNPs identified by NGS from strains MN7 and MN55 and were confirmed by Sanger sequencing. (D) The WT allele of <i>HSC20</i> was cloned in two independent plasmids, and these were used to transform strain MN7 and independent transformants were analyzed. This complementation test shows that the TS phenotype of MN7 is attributable to the recessive <i>hsc20-1</i> mutation.</p

Aneuploidy is generated during a-α sexual reproduction.

<p>Genotypic analysis of representative progeny from (A) XL280α crossed with JEC20<b>a</b>; (B) XL280α “<b>a</b>-α” self-sexual reproduction generated via expression of <i>SXI2</i><b>a</b>; and (C) <i>C. neoformans</i> var. <i>grubii</i> strain KN99α crossed with congenic strain KN99<b>a </b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001653#pbio.1001653-Nielsen2" target="_blank">[70]</a>.</p

Aneuploid strains are fluconazole resistance.

<p>(A) XL280 meiotic progeny were grown in YPD, diluted 10-fold in water, spotted, and incubated under the following conditions: YPD at 30°C for 2 d, YPD at 37°C for 2 d, and YPD plus 8 µg/mL fluconazole (FLC) at 30°C for 6 d. (B) Three aneuploid strains exhibited increased competitive fitness in competition assays. An XL280 strain marked with a <i>NAT</i> resistance marker integrated in the <i>SPO11</i> genetic locus was mixed together at equal abundance with XL280, MN55 (n+1⁹), MN77 (n+1¹⁰), or MN89 (n+1¹⁰). The mixtures were incubated on YPD and YPD plus 8 µg/mL fluconazole (FLC) at 30°C for 3 d. The cells were scraped from the plates, serially diluted, and plated on YPD and YPD plus NAT. The ratios of survival of each strain are shown for one representative experiment, and * represents significance at <i>p</i><0.005 from three independent experiments. We note that the <i>NAT</i> marker was neutral in this analysis and served to mark and detect the WT competitor cells. (C) The three aneuploid strains exhibited higher growth rates in the presence of fluconazole. Growth curves of the aneuploid strains were similar to the wild-type in rich media. However, in the presence of fluconazole the growth rate of the aneuploid strains was significantly higher than the wild-type.</p