Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.     

A unique cell wall synthetic response evoked by glucosamine determines pathogenicity-associated fungal cellular differentiation

The yeast-to-hypha transition is tightly associated with pathogenicity in many human pathogenic fungi, such as the model fungal pathogen Cryptococcus neoformans, which is responsible for approximately 180,000 deaths annually. In this pathogen, the yeast-to-hypha transition can be initiated by distinct stimuli: mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. However, it remains poorly understood how the signal specificity for Cryptococcus morphological transition by disparate stimuli is ensured. Here, by integrating temporal expression signature analysis and phenome-based clustering evaluation, we demonstrate that GlcN specifically triggers a unique cellular response, which acts as a critical determinant underlying the activation of GlcN-induced filamentation (GIF). This cellular response is defined by an unusually hyperactive cell wall synthesis that is highly ATP-consuming. A novel cell surface protein Gis1 was identified as the indicator molecule for the GlcN-induced cell wall response. The Mpk1-directed cell wall pathway critically bridges global cell wall gene induction and intracellular ATP supply, ensuring the Gis1-dependent cell wall response and the stimulus specificity of GIF. We further reveal that the ability of Mpk1 to coordinate the cell wall response and GIF activation is conserved in different Cryptococcus pathogens. Phosphoproteomics-based profiling together with genetic and phenotypic analysis revealed that the Mpk1 kinase mediates the regulatory specificity of GIF through a coordinated downstream regulatory network centered on Skn7 and Crz1. Overall, our findings discover an unprecedented and conserved cell wall biosynthesis-dependent fungal differentiation commitment mechanism, which enables the signal specificity of pathogenicity-related dimorphism induced by GlcN in Cryptococcus pathogens.     

Unisexual Reproduction Enhances Fungal Competitiveness by Promoting Habitat Exploration via Hyphal Growth and Sporulation

Unisexual reproduction is a novel homothallic sexual cycle recently discovered in both ascomycetous and basidiomycetous pathogenic fungi. It is a form of selfing that induces the yeast-to-hyphal dimorphic transition in isolates of the α mating type of the human fungal pathogen Cryptococcus neoformans. Unisexual reproduction may benefit the pathogen by facilitating sexual reproduction in the absence of the opposite a mating type and by generating infectious propagules called basidiospores. Here, we report an independent potential selective advantage of unisexual reproduction beyond genetic exchange and recombination. We competed a wild-type strain capable of undergoing unisexual reproduction with mutants defective in this developmental pathway and found that unisexual reproduction provides a considerable dispersal advantage through hyphal growth and sporulation. Our results show that unisexual reproduction may serve to facilitate access to both nutrients and potential mating partners and may provide a means to maintain the capacity for dimorphic transitions in the environment.     

Effect of Tetrandrine against Candida albicans Biofilms

Candida albicans is the most common human fungal pathogen and has a high propensity to develop biofilms that are resistant to traditional antifungal agents. In this study, we investigated the effect of tetrandrine (TET) on growth, biofilm formation and yeast-to-hypha transition of C. albicans. We characterized the inhibitory effect of TET on hyphal growth and addressed its possible mechanism of action. Treatment of TET at a low concentration without affecting fungal growth inhibited hyphal growth in both liquid and solid Spider media. Real-time RT-PCR revealed that TET down-regulated the expression of hypha-specific genes ECE1, ALS3 and HWP1, and abrogated the induction of EFG1 and RAS1, regulators of hyphal growth. Addition of cAMP restored the normal phenotype of the SC5314 strain. These results indicate that TET may inhibit hyphal growth through the Ras1p-cAMP-PKA pathway. In vivo, at a range of concentrations from 4 mg/L to 32 mg/L, TET prolonged the survival of C. albicans-infected Caenorhabditis elegans significantly. This study provides useful information for the development of new strategies to reduce the incidence of C. albicans biofilm-associated infections.     

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.     

αADα Hybrids of Cryptococcus neoformans: Evidence of Same-Sex Mating in Nature and Hybrid Fitness

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.     

Rad6p represses yeast-hypha morphogenesis in the human fungal pathogen Candida albicans

Rad6p plays important roles in post-replication DNA repair, chromatin organization, gene silencing and meiosis. In this study, we show that Rad6p also regulates yeast-hypha morphogenesis in the human pathogen Candida albicans. CaRAD6 gene and cDNAs were isolated and characterized revealing that the gene carries two 5'-proximal introns. CaRad6p shows a high degree of sequence similarity to Rad6 proteins from fungi to man (60-83% identity), and it suppresses the UV sensitivity and lack of induced mutagenesis displayed by a Saccharomyces cerevisiae rad6 mutant. In C. albicans, CaRAD6 expression is induced in response to UV, and CaRad6p depletion confers UV sensitivity, confirming that Rad6p serves a role in protecting this fungus against UV damage. CaRAD6 overexpression inhibits hyphal development, whereas CaRad6p depletion enhances hyphal growth. Also, CaRAD6 mRNA levels decrease during the yeast-hypha transition. These effects are dependent on Efg1p, but not Cph1p, indicating that CaRad6p acts specifically through the Efg1p morphogenetic signalling pathway to repress yeast-hypha morphogenesis.     

Unisexual Reproduction of Cryptococcus gattii

Cryptococcus gattii is a basidiomycetous human fungal pathogen that typically causes infection in tropical and subtropical regions and is responsible for an ongoing outbreak in immunocompetent individuals on Vancouver Island and in the Pacific Northwest of the US. Pathogenesis of this species may be linked to its sexual cycle that generates infectious propagules called basidiospores. A marked predominance of only one mating type (α) in clinical and environmental isolates suggests that a-α opposite-sex reproduction may be infrequent or geographically restricted, raising the possibility of an alternative unisexual cycle involving cells of only α mating type, as discovered previously in the related pathogenic species Cryptococcus neoformans. Here we report observation of hallmark features of unisexual reproduction in a clinical isolate of C. gattii (isolate 97/433) and describe genetic and environmental factors conducive to this sexual cycle. Our results are consistent with population genetic evidence of recombination in the largely unisexual populations of C. gattii and provide a useful genetic model for understanding how novel modes of sexual reproduction may contribute to evolution and virulence in this species.     

Characterizing the role of the microtubule binding protein Bim1 in Cryptococcus neoformans

During sexual development the human fungal pathogen Cryptococcus neoformans undergoes a developmental transition from yeast-form growth to filamentous growth. This transition requires cellular restructuring to form a filamentous dikaryon. Dikaryotic growth also requires tightly controlled nuclear migration to ensure faithful replication and dissemination of genetic material to spore progeny. Although the gross morphological changes that take place during dikaryotic growth are largely known, the molecular underpinnings that control this process are uncharacterized. Here we identify and characterize a C. neoformans homolog of the Saccharomyces cerevisiae BIM1 gene, and establish the importance of BIM1 for proper filamentous growth of C. neoformans. Deletion of BIM1 leads to truncated sexual development filaments, a severe defect in diploid formation, and a block in monokaryotic fruiting. Our findings lead to a model consistent with a critical role for BIM1 in both filament integrity and nuclear congression that is mediated through the microtubule cytoskeleton.     

Sex-Induced Silencing Operates During Opposite-Sex and Unisexual Reproduction in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from yeast to hyphae during a-α opposite-sex mating and α-α unisexual reproduction (same-sex mating). Infectious spores are generated during both processes. We previously identified a sex-induced silencing (SIS) pathway in the C. neoformans serotype A var. grubii lineage, in which tandem transgene arrays trigger RNAi-dependent gene silencing at a high frequency during a-α opposite-sex mating, but at an ∼250-fold lower frequency during asexual mitotic vegetative growth. Here we report that SIS also operates during α-α unisexual reproduction. A self-fertile strain containing either SXI2a-URA5 or NEO-URA5 transgene arrays exhibited an elevated silencing frequency during solo and unisexual mating compared with mitotic vegetative growth. We also found that SIS operates at a similar efficiency on transgene arrays of the same copy number during either α-α unisexual reproduction or a-α opposite-sex mating. URA5-derived small RNAs were detected in the silenced progeny of α-α unisexual reproduction and RNAi core components were required, providing evidence that SIS induced by same-sex mating is also mediated by RNAi via sequence-specific small RNAs. In addition, our data show that the SIS RNAi pathway also operates to defend the genome via squelching transposon activity during same-sex mating as it does during opposite-sex mating. Taken together, our results confirm that SIS is conserved between the divergent C. neoformans serotype A and serotype D cryptic sibling species.     

Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C,Focal Adhesion Kinase,and Ezrin to Promote Traversal across the Blood-Brain Barrier

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.     

The Golgi GDPase of the fungal pathogen Candida albicans affects morphogenesis,glycosylation, and cell wall properties

Cell wall mannoproteins are largely responsible for the adhesive properties and immunomodulation ability of the fungal pathogen Candida albicans. The outer chain extension of yeast mannoproteins occurs in the lumen of the Golgi apparatus. GDP-mannose must first be transported from the cytosol into the Golgi lumen, where mannose is transferred to mannans. GDP is hydrolyzed by a GDPase, encoded by GDA1, to GMP, which then exits the Golgi lumen in a coupled, equimolar exchange with cytosolic GDP-mannose. We isolated and disrupted the C. albicans homologue of the Saccharomyces cerevisiae GDA1 gene in order to investigate its role in protein mannosylation and pathogenesis. CaGda1p shares four apyrase conserved regions with other nucleoside diphosphatases. Membranes prepared from the C. albicans disrupted gda1/gda1 strain had a 90% decrease in the ability to hydrolyze GDP compared to wild type. The gda1/gda1 mutants showed a severe defect in O-mannosylation and reduced cell wall phosphate content. Other cell wall-related phenotypes are present, such as elevated chitin levels and increased susceptibility to attack by β-1,3-glucanases. Our results show that the C. albicans organism contains β-mannose at their nonreducing end, differing from S. cerevisiae, which has only α-linked mannose residues in its O-glycans. Mutants lacking both alleles of GDA1 grow at the same rate as the wild type but are partially blocked in hyphal formation in Lee solid medium and during induction in liquid by changes in temperature and pH. However, the mutants still form normal hyphae in the presence of serum and N-acetylglucosamine and do not change their adherence to HeLa cells. Taken together, our data are in agreement with the hypothesis that several pathways regulate the yeast-hypha transition. Gda1/gda1 cells offer a model for discriminating among them.     

Novel Mechanism Coupling Cyclic AMP-Protein Kinase A Signaling and Golgi Trafficking via Gyp1 Phosphorylation in Polarized Growth

The cyclic AMP (cAMP)-protein kinase A (PKA) signaling activates virulence expression during hyphal development in the fungal human pathogen Candida albicans. The hyphal growth is characterized by Golgi polarization toward the hyphal tips, which is thought to enhance directional vesicle transport. However, how the hypha-induction signal regulates Golgi polarization is unknown. Gyp1, a Golgi-associated protein and the first GTPase-activating protein (GAP) in the Rab GAP cascade, critically regulates membrane trafficking from the endoplasmic reticulum to the plasma membrane. Here, we report a novel pathway by which the cAMP-PKA signaling triggers Golgi polarization during hyphal growth. We demonstrate that Gyp1 plays a crucial role in actin-dependent Golgi polarization. Hyphal induction activates PKA, which in turn phosphorylates Gyp1. Phosphomimetic mutation of four PKA sites identified by mass spectrometry (Gyp1^4E) caused strong Gyp1 polarization to hyphal tips, whereas nonphosphorylatable mutations (Gyp1^4A) abolished it. Gyp1^4E exhibited enhanced association with the actin motor Myo2, while Gyp1^4A showed the opposite effect, providing a possible mechanism for Golgi polarization. A GAP-dead Gyp1 (Gyp1^R292K) showed strong polarization similar to that seen with Gyp1^4E, indicating a role for the GAP activity. Mutating the PKA sites on Gyp1 also impaired the recruitment of a late Golgi marker, Sec7. Furthermore, proper PKA phosphorylation and GAP activity of Gyp1 are required for virulence in mice. We propose that the cAMP-PKA signaling directly targets Gyp1 to promote Golgi polarization in the yeast-to-hypha transition, an event crucial for C. albicans infection.