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

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

Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen <i>Cryptococcus neoformans</i>

<div><p>Calcineurin is a highly conserved Ca²⁺/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca²⁺ signaling responses. In <i>Cryptococcus neoformans</i>, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in <i>Saccharomyces cerevisiae</i> and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in <i>cna1</i>Δ and <i>crz1</i>Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate <i>crz1</i>Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in <i>C</i>. <i>neoformans</i> appears to be adapted to promote fungal virulence.</p></div

<i>C. amylolentus</i> has a tetrapolar mating system.

<p>(A) In a bipolar mating system, haploid <b>a</b> and α cells fuse to form a diploid <b>a</b>/α cell. Sex culminates in meiosis, which gives rise to four meiotic progeny, 2 <b>a</b> and 2 α. The <b>a</b> progeny can mate with the α parent (50%) while the α progeny can mate with the <b>a</b> parent (50%). In a tetrapolar mating system, haploid A1B1 and A2B2 cells fuse to form a dikaryon/diploid A1B1/A2B2. Meiosis then results in the production of four haploid meiotic progeny: A1B1 can mate with the A2B2 parent and progeny (25%), A2B2 can mate with the A1B1 parent and progeny (25%), and A1B2 and A2B1 are recombinants (50%) that are sterile with either parent but interfertile with one another. (B) An example of a RAPD and genotyping marker analysis on four progeny and the two parental strains that represent the different gentoypes in a tetrapolar mating system (1 = F1S2 #3 (A1B1), 2 = F1S2 #13 (A2B2), 3 = F2 #1 (A2B1), 4 = F1S2 #10 (A1B2), 5 = CBS6039 (A1B1), and 6 = CBS6273 (A2B2)). (C) Results of mating assays of all possible combinations among the four mating types. Mating was performed by mixing strains on V8 plate (pH = 5). (“−” indicates lack of sexual reproduction and “+” indicates sexual reproduction occurs). (D) Microscopic images of hyphae and spore chains generated during <i>C. amylolentus</i> mating assays described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002528#pgen-1002528-g009" target="_blank">Figure 9C</a> (the mating-type of each strain is indicated in parenthesis). Dikaryotic hyphae and spore chains were produced in matings between CBS6039 (A1B1) and CBS6273 (A2B2) and between F1 set2 #10 (A1B2) and F2 #1 (A2B1). Monokaryotic hyphae were produced in all of the other mating combinations, including individual strains grown in the absence of a mating partner.</p

Model for the evolution of the mating-type locus in the pathogenic <i>Cryptococcus</i> species.

<p>The physically unlinked ancestral tetrapolar HD and P/R loci contained both homeodomain genes and the pheromone/receptor genes respectively. Additional genes were acquired into both loci, expanding the <i>MAT</i>-specific region. A translocation event occurred, likely between chromosomes 4 and 5 of <i>Cryptococcus</i>, resulting in the formation of a transient tripolar intermediate and one of the HD genes was lost. The hypothetical genes (grey arrows) relocated, likely through a translocation event, to the telomeric ends of chromosome 4. The unstable tripolar intermediate later collapsed to a bipolar state. The fused loci were subjected to further gene rearrangement and gene conversion events, which led to the formation of the bipolar alleles of the pathogenic <i>Cryptococcus</i> species. White arrows indicate the five genes most recently acquired into <i>Cryptococcus MAT</i> locus and black arrows are <i>MAT</i>-specific genes present in the pathogenic <i>Cryptococcus</i> species.</p

Genes regulated by the calcineurin-Crz1 pathway feature three motifs.

<p><b>(A)</b> DNA motifs generated by MEME from promoter analysis of the 102 target genes. <b>(B)</b> DNA motif generated by DREME from promoter analysis of <i>CHS6</i> (CNAG_00546), and three other genes (CNAG_00588, CNAG_04891, and CNAG_00407). <b>(C)</b> Comparison of the presence or absence of the motifs against the phenotypic analyses of the target gene deletion mutants. CaCl₂ = 0.5 M calcium chloride; CR = 1% Congo red; CFW = 5 mg/mL calcofluor white.</p

Summary of mating abilities and genotypes at the <i>MAT</i> genes of F1 set 2 and F2 progeny.

<p>Bold: fertile with parents;</p><p>Bold and Italics: fertile with siblings from F1 set 1 and F2 progeny;</p><p>Underlined genotypes indicate intra-<i>MAT</i> (A or B locus) recombinant progeny;</p>1<p>: “a” represents allele from A1B1 parent CBS6039; “b” represents allele from A2B2 parent CBS6273.</p

Phylogenetic patterns of four <i>C. amylolentus MAT</i> genes.

<p>The phylogenetic relationships of <i>C. amylolentus</i> to the pathogenic <i>Cryptococcus</i> species and neighboring taxa based on four genes, <i>GEF1</i>, <i>CID1</i>, <i>SXI1</i>, and <i>SXI2</i>, are shown. <i>GEF1</i> and <i>CID1</i> display a species-specific phylogeny and the <i>SXI1</i> and <i>SXI2</i> alleles are very diverged from the pathogenic <i>Cryptococcus</i> species. The trees were constructed using the Neighbor-Joining method implemented in the software MEGA4. Bootstrap values on tree branches were calculated from 500 replicates. (α) indicates strains with the <i>MAT</i>α locus, and (<b>a</b>) indicates strains with the <i>MAT</i><b>a</b> locus.</p

<i>T. wingfieldii MAT</i> loci and chromosomal locations.

<p>(A) Six fosmids were analyzed to generate the assembly for <i>T. wingfieldii</i>. The <i>MAT</i> gene probes used to probe the <i>T. wingfieldii</i> library are indicated in blue. The HD (<i>B</i>) and P/R (<i>A</i>) loci are embedded within assemblies that span 40 and 70 kb respectively. Grey arrows indicate genes that either flank <i>MAT</i> or are hypothetical genes, black arrows are <i>Cryptococcus MAT</i>-specific genes, and yellow indicates the genes most recently acquired into the <i>Cryptococcus MAT</i> locus. Scale bar = 10 kb. (B) Chromosomes from <i>T. wingfieldii</i> were separated using PFGE, followed by Southern hybridization using three <i>MAT</i>-specific probes, two from the HD locus and one from the P/R locus. Arrows depict hybridization of HD genes to an ∼1 Mb chromosome distinct from hybridization of the P/R genes to an ∼1.1 Mb chromosome.</p