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

Deletion of Cryptococcus neoformans AIF Ortholog Promotes Chromosome Aneuploidy and Fluconazole-Resistance in a Metacaspase-Independent Manner

Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLCR) in vitro and in vivo. FLCR isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLCR subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance

An Antifungal Combination Matrix Identifies a Rich Pool of Adjuvant Molecules that Enhance Drug Activity against Diverse Fungal Pathogens

There is an urgent need to identify new treatments for fungal infections. By combining sub-lethal concentrations of the known antifungals fluconazole, caspofungin, amphotericin B, terbinafine, benomyl, and cyprodinil with ∼3,600 compounds in diverse fungal species, we generated a deep reservoir of chemical-chemical interactions termed the Antifungal Combinations Matrix (ACM). Follow-up susceptibility testing against a fluconazole-resistant isolate of C. albicans unveiled ACM combinations capable of potentiating fluconazole in this clinical strain. We used chemical genetics to elucidate the mode of action of the antimycobacterial drug clofazimine, a compound with unreported antifungal activity that synergized with several antifungals. Clofazimine induces a cell membrane stress for which the Pkc1 signaling pathway is required for tolerance. Additional tests against additional fungal pathogens, including Aspergillus fumigatus, highlighted that clofazimine exhibits efficacy as a combination agent against multiple fungi. Thus, the ACM is a rich reservoir of chemical combinations with therapeutic potential against diverse fungal pathogens

Systematic functional profiling of transcription factor networks in <em>Cryptococcus neoformans</em>

International audienceCryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and ∼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens

Analysis of the genome and transcriptome of [i]Cryptococcus neoformans[/i] var.[i] grubii[/i] 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

miscRNAs in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Two examples of a miscRNA as visualized through Artemis. The coverage of the plus stand is represented by the black curve. The coverage of the minus strand is represented by the blue curve. These results were obtained when cells grown in low glucose and nitrogen medium (starvation medium) underwent strand-specific sequencing. F1, F2, and F3 stand for 5′ to 3′ frames 1, 2, and 3, respectively. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2 and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. <b>B.</b> Schematic representation of the positions of the miscRNAs in the <i>C. neoformans</i> var. <i>grubii</i> genome as compared to coding sequences. The numbers of miscRNAs at each position is indicated. The number of miscRNAs in the antisense strand of other miscRNAs is indicated between brackets.</p

Antisense/sense transcription in <i>C. neoformans</i> var. <i>grubii</i>.

<p><b>A.</b> Comparison of sense/antisense transcription when an antisense transcript is present. Strand-specific data obtained from cells grown on YPD is shown. The BaseMean values represent the normalized reads count for each transcript and measure the level of sense transcription (x axis) and antisense transcription (y axis) as calculated by DESeq <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004261#pgen.1004261-Anders1" target="_blank">[126]</a>. Outliers with a BaseMean above 12,000 were not represented. <b>B.</b> Example of differential expression of miscRNA antisense of a coding gene as observed through Artemis. The red curve represents the non-strand-specific coverage observed when cells were grown in YPD to stationary phase at 30°C (condition 1); the green curve shows the non-strand-specific coverage observed when the cells were grown in YPD to log phase at 30°C (condition 2). F1, F2, and F3 stand for 5′ to 3′ frames 1, 2, and 3, respectively. F4, F5, and F6 stand for 3′ to 5′ frames 1, 2, and 3, respectively. The small black vertical bars indicate the position of the stop codons for each frame. <b>C.</b> Northern blot obtained after hybridization with strand-specific probes. RNA was extracted from cells growing in YPD (2×10⁸ cells/mL) at 30°C (condition 1), YPD (5×10⁷ cells/mL) at 30°C (condition 2), YPD with 0.01% SDS (5×10⁷ cells/mL) at 30°C (condition 3), YPD with 10 mg/mL fluconazole (5×10⁷ cells/mL) at 30°C (condition 4), YPD (5×10⁷ cells/mL) at 37°C (condition 5), and YP galactose (2×10⁸ cells/mL) at 30°C (condition 6) in duplicate. Then, 5 µg RNA were loaded on a denaturing electrophoresis agarose gel, electrophoresed, and transferred to a nylon membrane. The horizontal black line represents the position of the probes.</p