Yen-Ping Hsueh

Spores as Infectious Propagules of Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (∼95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.

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.

Magnificent seven : roles of G protein-coupled receptors in extracellular sensing in fungi

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks. This review highlights recent research advances in fungal GPCRs, including classification, extracellular sensing, and G protein-signaling regulation. The involvement of GPCRs in pheromone and nutrient sensing has been studied extensively over the past decade. Following recent advances in fungal genome sequencing projects, a panoply of GPCR candidates has been revealed and some have been documented to play key roles sensing diverse extracellular signals, such as pheromones, sugars, amino acids, nitrogen sources, and even photons. Identification and deorphanization of additional putative GPCRs may require the development of new research tools. Here, we compare research on GPCRs in fungi with information derived from mammalian systems to provide a useful road map on how to better understand ligand-GPCR-G protein interactions in general. We also emphasize the utility of yeast as a discovery tool for systemic studies of GPCRs from other organisms.

Sex-induced silencing defends the genome of Cryptococcus neoformans via RNAi

Cosuppression is a silencing phenomenon triggered by the introduction of homologous DNA sequences into the genomes of organisms as diverse as plants, fungi, flies, and nematodes. Here we report sex-induced silencing (SIS), which is triggered by tandem integration of a transgene array in the human fungal pathogen Cryptococcus neoformans. A SXI2a-URA5 transgene array was found to be post-transcriptionally silenced during sexual reproduction. More than half of the progeny that inherited the SXI2a-URA5 transgene became uracil-auxotrophic due to silencing of the URA5 gene. In vegetative mitotic growth, silencing of this transgene array occurred at an ∼250-fold lower frequency, indicating that silencing is induced during the sexual cycle. Central components of the RNAi pathway-including genes encoding Argonaute, Dicer, and an RNA-dependent RNA polymerase-are all required for both meiotic and mitotic transgene silencing. URA5-derived ∼22-nucleotide (nt) small RNAs accumulated in the silenced isolates, suggesting that SIS is mediated by RNAi via sequence-specific small RNAs. Through deep sequencing of the small RNA population in C. neoformans, we also identified abundant small RNAs mapping to repetitive transposable elements, and these small RNAs were absent in rdp1 mutant strains. Furthermore, a group of retrotransposons was highly expressed during mating of rdp1 mutant strains, and an increased transposition/mutation rate was detected in their progeny, indicating that the RNAi pathway squelches transposon activity during the sexual cycle. Interestingly, Ago1, Dcr1, Dcr2, and Rdp1 are translationally induced in mating cells, and Ago1, Dcr1, and Dcr2 localize to processing bodies (P bodies), whereas Rdp1 appears to be nuclear, providing mechanistic insights into the elevated silencing efficiency during sexual reproduction. We hypothesize that the SIS RNAi pathway operates to defend the genome during sexual development.

Orchestration of sexual reproduction and virulence by the fungal mating-type locus.

The mating-type locus (MAT) orchestrates sexual reproduction in fungi. Sexual reproduction is related not only to fitness of an organism, but also correlated with virulence in certain pathogens. In the dandruff-associated fungus Malassesia globosa, although the sexual cycle remains to be discovered, whole genome analysis has led to the hypothesis that mating may occur on host skin. Furthermore, the MAT locus of M. globosa and U. hordei provides evidence that transitions between tetrapolar and bipolar systems have independently occurred. These results, together with studies recapitulating the ancestral tetrapolar mating system in Cryptococcus and the structure of MAT in related smut fungi, have furthered understanding on transitions between different mating systems and the evolution of MAT in the Basidiomycota.

Nematode-trapping fungi eavesdrop on nematode pheromones.

The recognition of molecular patterns associated with specific pathogens or food sources is fundamental to ecology and plays a major role in the evolution of predator-prey relationships. Recent studies showed that nematodes produce an evolutionarily highly conserved family of small molecules, the ascarosides, which serve essential functions in regulating nematode development and behavior. Here, we show that nematophagous fungi, natural predators of soil-dwelling nematodes, can detect and respond to ascarosides. Nematophagous fungi use specialized trapping devices to catch and consume nematodes, and previous studies demonstrated that most fungal species do not produce traps constitutively but rather initiate trap formation in response to their prey. We found that ascarosides, which are constitutively secreted by many species of soil-dwelling nematodes, represent a conserved molecular pattern used by nematophagous fungi to detect prey and trigger trap formation. Ascaroside-induced morphogenesis is conserved in several closely related species of nematophagous fungi and occurs only under nutrient-deprived conditions. Our results demonstrate that microbial predators eavesdrop on chemical communication among their metazoan prey to regulate morphogenesis, providing a striking example of predator-prey coevolution. We anticipate that these findings will have broader implications for understanding other interkingdom interactions involving nematodes, which are found in almost any ecological niche on Earth.

The RGS protein Crg2 regulates both pheromone and cAMP signalling in Cryptococcus neoformans

G proteins orchestrate critical cellular functions by transducing extracellular signals into internal signals and controlling cellular responses to environmental cues. G proteins typically function as switches that are activated by G protein‐coupled receptors (GPCRs) and negatively controlled by regulator of G protein signalling (RGS) proteins. In the human fungal pathogen Cryptococcus neoformans, three G protein α subunits (Gpa1, Gpa2 and Gpa3) have been identified. In a previous study, we identified the RGS protein Crg2 involved in regulating the pheromone response pathway through Gpa2 and Gpa3. In this study, a role for Crg2 was established in the Gpa1‐cAMP signalling pathway that governs mating and virulence. We show that Crg2 physically interacts with Gpa1 and crg2 mutations increase cAMP production. crg2 mutations also enhance mating filament hyphae production, but reduce cell–cell fusion and sporulation efficiency during mating. Although crg2 mutations and the Gpa1 dominant active allele GPA1Q284L enhanced melanin production under normally repressive conditions, virulence was attenuated in a murine model. We conclude that Crg2 participates in controlling both Gpa1‐cAMP‐virulence and pheromone‐mating signalling cascades and hypothesize it may serve as a molecular interface between these two central signalling conduits.

A constitutively active GPCR governs morphogenic transitions in Cryptococcus neoformans

Sex in fungi is driven by peptide pheromones sensed through seven‐transmembrane pheromone receptors. In Cryptococcus neoformans, sexual reproduction occurs through an outcrossing/heterothallic a‐ sexual cycle or an inbreeding/homothallic – unisexual mating process. Pheromone receptors encoded by the mating‐type locus (MAT) mediate reciprocal pheromone sensing during opposite‐sex mating and contribute to but are not essential for unisexual mating. A pheromone receptor‐like gene, CPR2, was discovered that is not encoded by MAT and whose expression is induced during a‐ mating. cpr2 mutants are fertile but have a fusion defect and produce abnormal hyphal structures, whereas CPR2 overexpression elicits unisexual reproduction. When heterologously expressed in Saccharomyces cerevisiae, Cpr2 activates pheromone responses in the absence of any ligand. This constitutive activity results from an unconventional residue, Leu222, in place of a conserved proline in transmembrane domain six; a Cpr2L222P mutant is no longer constitutively active. Cpr2 engages the same G‐protein activated signalling cascade as the Ste3a/α pheromone receptors, and thereby competes for pathway activation. This study established a new paradigm in which a naturally occurring constitutively active G protein‐coupled receptor governs morphogenesis in fungi.

Transitions in Sexuality: Recapitulation of an Ancestral Tri- and Tetrapolar Mating System in Cryptococcus neoformans

ABSTRACT Sex is orchestrated by the mating-type locus (MAT) in fungi and by sex chromosomes in plants and animals. In fungi, two patterns of sexuality occur: bipolar with a single, typically biallelic sex determinant that promotes inbreeding, and tetrapolar with two unlinked, often multiallelic sex determinants that restrict inbreeding. Multiallelism in either bipolar or tetrapolar mating systems promotes outcrossing. Cryptococcus neoformans is a pathogenic bipolar yeast with two unusually large MAT alleles (a/α) spanning >100 kb, ∼100-fold larger than many other fungal MAT loci. Based on comparative genomic analysis, this unusual MAT locus is hypothesized to have evolved from an ancestral tetrapolar system. In this model, the unlinked homeodomain (HD) transcription factor and pheromone/receptor tetrapolar loci acquired additional sex-related genes and then fused via chromosomal translocation, forming an intermediate transitional mating system (which we term tripolar), which then underwent recombination and gene conversion to fashion the extant bipolar MAT alleles. To experimentally validate this model, C. neoformans was engineered to have a tetrapolar mating system by relocating the MAT SXI1α and SXI2a HD genes to an unlinked genomic locale. Genetic and molecular analyses revealed that this modified organism could complete a tetrapolar sexual cycle. Analysis of progeny generated from bipolar, tripolar, and tetrapolar crosses provides direct experimental evidence that the tripolar state confers decreased fertility and therefore may represent an unstable evolutionary intermediate. These findings illustrate how transitions between outcrossing and inbreeding preference occur by involving sex determinant linkage and collapse from multiallelic to biallelic sex determination, providing insights into both fungal sex evolution and early steps in sex chromosome evolution.

Transmission of Hypervirulence Traits via Sexual Reproduction within and between Lineages of the Human Fungal Pathogen Cryptococcus gattii

Since 1999 a lineage of the pathogen Cryptococcus gattii has been infecting humans and other animals in Canada and the Pacific Northwest of the USA. It is now the largest outbreak of a life-threatening fungal infection in a healthy population in recorded history. The high virulence of outbreak strains is closely linked to the ability of the pathogen to undergo rapid mitochondrial tubularisation and proliferation following engulfment by host phagocytes. Most outbreaks spread by geographic expansion across suitable niches, but it is known that genetic re-assortment and hybridisation can also lead to rapid range and host expansion. In the context of C. gattii, however, the likelihood of virulence traits associated with the outbreak lineages spreading to other lineages via genetic exchange is currently unknown. Here we address this question by conducting outgroup crosses between distantly related C. gattii lineages (VGII and VGIII) and ingroup crosses between isolates from the same molecular type (VGII). Systematic phenotypic characterisation shows that virulence traits are transmitted to outgroups infrequently, but readily inherited during ingroup crosses. In addition, we observed higher levels of biparental (as opposed to uniparental) mitochondrial inheritance during VGII ingroup sexual mating in this species and provide evidence for mitochondrial recombination following mating. Taken together, our data suggest that hypervirulence can spread among the C. gattii lineages VGII and VGIII, potentially creating novel hypervirulent genotypes, and that current models of uniparental mitochondrial inheritance in the Cryptococcus genus may not be universal.