Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen that causes meningitis in immunocompromised patients. Its growth is sensitive to the immunosuppressants FK506 and cyclosporin, which inhibit the Ca2+- calmodulin-activated protein phosphatase calcineurin. Calcineurin is required for growth at 37 degrees C and virulence of C.neoformans. We found that calcineurin is also required for mating. FK506 blocks mating of C.neoformans via FKBP12-dependent inhibition of calcineurin, and mutants lacking calcineurin are bilaterally sterile. Calcineurin is not essential for the initial fusion event, but is required for hyphal elongation and survival of the heterokaryon produced by cell fusion. It is also required for hyphal elongation in diploid strains and during asexual haploid fruiting of MATalpha cells in response to nitrogen limitation. Because mating and haploid fruiting produce infectious basidiospores, our studies suggest a second link between calcineurin and virulence of C.neoformans. Calcine urin regulates filamentation and 37 degrees C growth via distinct pathways. Together with studies revealing that calcineurin mediates neurite extension and neutrophil migration in mammals, our findings indicate that calcineurin plays a conserved role in the control of cell morphology.     

A homolog of Ste6, the a-factor transporter in Saccharomyces cerevisiae, is required for mating but not for monokaryotic fruiting in Cryptococcus neoformans

Fungal pheromones function during the initial recognition stage of the mating process. One type of peptide pheromone identified in ascomycetes and basidiomycetes terminates in a conserved CAAX motif and requires extensive posttranslational modifications to become mature and active. A well-studied representative is the a-factor of Saccharomyces cerevisiae. Unlike the typical secretory pathway utilized by most peptides, an alternative mechanism involving the ATP-binding cassette transporter Ste6 is used for the export of mature a-factor. Cryptococcus neoformans, a bipolar human pathogenic basidiomycete, produces CAAX motif-containing lipopeptide pheromones in both MATa and MATalpha cells. Virulence studies with a congenic pair of C. neoformans serotype D strains have shown that MATalpha cells are more virulent than MATa cells. Characterization of the MATalpha pheromones indicated that an autocrine signaling loop may contribute to the differentiation and virulence of MATalpha cells. To further address the role of pheromones in the signaling loop, we identified a STE6 homolog in the C. neoformans genome and determined its function by gene disruption. The ste6 mutants in either mating-type background showed partially impaired mating functions, and mating was completely abolished in a bilateral mutant cross. Surprisingly, the MATalpha ste6 mutant does not exhibit a defect in monokaryotic fruiting, suggesting that the activation of the autocrine signaling loop by the pheromone is via a Ste6-independent mechanism. MFalpha pheromone itself is essential for this process and could induce the signaling response intracellularly in MATalpha cells. Our data demonstrate that Ste6 is evolutionarily conserved for mating and is not required for monokaryotic fruiting in C. neoformans.     

Good fungi gone bad: the corruption of calcineurin

Calcineurin is a Ca(2+)/calmodulin-activated protein phosphatase that is conserved in eukaryotes, from yeast to humans, and is the conserved target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. Genetic studies in yeast and fungi established the molecular basis of calcineurin inhibition by the cyclophilin A-CsA and FKBP12-FK506 complexes. Calcineurin also functions in fungi to control a myriad of physiological processes including cell cycle progression, cation homeostasis, and morphogenesis. Recent investigations into the molecular mechanisms of pathogenesis in Candida albicans and Cryptococcus neoformans, two fungi that cause life-threatening infections in humans, have revealed an essential role for calcineurin in morphogenesis, virulence, and antifungal drug action. Novel non-immunosuppressive analogs of the calcineurin inhibitors CsA and FK506 that retain antifungal activity have been identified and hold promise as candidate antifungal drugs. In addition, comparisons of calcineurin function in both fungi and humans may identify fungal-specific components of calcineurin-signaling pathways that could be targeted for therapy, as well as conserved elements of calcium signaling events.     

Signal transduction cascades regulating mating, filamentation, and virulence in Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycetous fungal pathogen that infects the central nervous system. The organism has a defined sexual cycle involving mating between haploid MATalpha and MATa cells. Recent studies have revealed signaling cascades that coordinately regulate differentiation and virulence of C. neoformans. One signaling cascade involves a conserved G-protein alpha subunit and cAMP, and senses nutrients during mating and virulence. The second is a conserved mitogen activated protein (MAP) kinase cascade that senses pheromone during mating, and also regulates haploid fruiting and virulence. Interestingly, some of the MAP kinase components are encoded by the MAT locus itself, which may explain the unique association of the MATalpha locus with physiology and virulence.     

Coping with stress: calmodulin and calcineurin in model and pathogenic fungi

Calcium signaling via calmodulin and calcineurin is critical for the regulation of stress responses in fungi. The functions of calmodulin and calcineurin are largely conserved among pathogenic fungi and model fungi, however, the mechanisms of action have diverged. Saccharomyces cerevisiae is an excellent model for understanding the framework of calcium-mediated signal transduction pathways, and considerable progress has been made in understanding the details of how Ca(2+)-calmodulin and calcineurin control adaptation to environmental stress. Studies using the divergent human pathogenic fungi Candida albicans and Cryptococcus neoformans reveal that calcineurin is critical for virulence, yet it acts via distinct mechanisms in each fungus. These differences in function may reflect the requirements of each pathogen to survive inside the host, and illustrate that studies must be conducted in each organism in order to elucidate the details of the molecular mechanisms of calmodulin and calcineurin-mediated signaling pathways.     

A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans

Pathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host. This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species. One such pathway, whose components include Ras proteins and the PAK kinase Ste20, allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature. Previously, we found that Ras1 signalling is required for differentiation, thermotolerance and pathogenesis in C. neoformans. We show here that the guanine nucleotide exchange factor Cdc24 is a Ras1 effector in C. neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease. In addition, we provide evidence that the Ras1-Cdc24 signalling cascade functions specifically through one of the three Cdc42/Rac1 homologues in C. neoformans. In conclusion, our studies illustrate how components of conserved signalling cascades can be specialized for different downstream functions, such as pathogenesis.     

Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade

Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin of new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus Cryptococcus. Previous studies revealed that two PKA catalytic subunits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the Cryptococcus neoformans var. grubii serotype A lineage compared to divergent C. neoformans var. neoformans serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species Cryptococcus gattii by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens.     

Canonical heterotrimeric G proteins regulating mating and virulence of Cryptococcus neoformans

Perturbation of pheromone signaling modulates not only mating but also virulence in Cryptococcus neoformans, an opportunistic human pathogen known to encode three Galpha, one Gbeta, and two Ggamma subunit proteins. We have found that Galphas Gpa2 and Gpa3 exhibit shared and distinct roles in regulating pheromone responses and mating. Gpa2 interacted with the pheromone receptor homolog Ste3alpha, Gbeta subunit Gpb1, and RGS protein Crg1. Crg1 also exhibited in vitro GAP activity toward Gpa2. These findings suggest that Gpa2 regulates mating through a conserved signaling mechanism. Moreover, we found that Ggammas Gpg1 and Gpg2 both regulate pheromone responses and mating. gpg1 mutants were attenuated in mating, and gpg2 mutants were sterile. Finally, although gpa2, gpa3, gpg1, gpg2, and gpg1 gpg2 mutants were fully virulent, gpa2 gpa3 mutants were attenuated for virulence in a murine model. Our study reveals a conserved but distinct signaling mechanism by two Galpha, one Gbeta, and two Ggamma proteins for pheromone responses, mating, and virulence in Cryptococcus neoformans, and it also reiterates that the link between mating and virulence is not due to mating per se but rather to certain mating-pathway components that encode additional functions promoting virulence.     

Calcium- and calcineurin-independent roles for calmodulin in Cryptococcus neoformans morphogenesis and high-temperature growth

The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37 degrees C. One insertion mutant, cam1-ts, that displayed a growth defect at 37 degrees C and hypersensitivity to the calcineurin inhibitor FK506 at 25 degrees C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3' untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37 degrees C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Delta diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37 degrees C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca2+-binding-defective allele of CAM1 complemented the 37 degrees C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.     

Cryptococcus neoformans mediator protein Ssn8 negatively regulates diverse physiological processes and is required for virulence

Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans.     

Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway

The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress response, and maintenance of cell wall integrity. Here we characterized a Cryptococcus neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence.     

The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high-temperature stress responses in Cryptococcus neoformans

Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The cts1Δ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurin-dependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.     

Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.     

Mating differentiation in Cryptococcus neoformans is negatively regulated by the Crk1 protein kinase

Cryptococcus neoformans is a heterothallic basidiomycete that grows vegetatively as yeast and filamentous hyphae are produced in the sexual state. Previous studies have shown that C. neoformans Cwc1 and Cwc2 are two central photoregulators which form a complex to inhibit the production of sexual filaments upon light treatment. To reveal the detailed regulatory mechanisms, a genome wide mutagenesis screen was conducted and components in the Cwc1/Cwc2 complex mediated pathway have been identified. In this study, one suppressor mutant, DJ22, is characterized and T-DNA is found to disrupt the C. neoformans CRK1 gene, a homologue of Saccharomyces cerevisiae IME2 and Ustilago maydis crk1. Ime2 is a meiosis-specific gene with the conserved Ser/Thr kinase domain and TXY dual phosphorylation site. Consistent with the findings of other suppressors in our screen, C. neoformans Crk1 plays a negative role in the mating process. Dikaryotic filaments, basidia, and basidiospores are produced earlier in the crk1 mutant crosses and mating efficiency is also increased. Artificial elevation of the CRK1 mRNA level inhibits mating. Interestingly, monokaryotic fruiting is defective both in the MATα crk1 mutant and CRK1 overexpression strains. Our studies demonstrate that C. neoformans CRK1 gene functions as a negative regulator in the mating differentiation.     

The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.     

Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene

Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.     

Signal transduction cascades regulating fungal development and virulence.

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.     

Signal Transduction Cascades Regulating Fungal Development and Virulence

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.     

Conserved elements of the RAM signaling pathway establish cell polarity in the basidiomycete Cryptococcus neoformans in a divergent fashion from other fungi

In eukaryotes the complex processes of development, differentiation, and proliferation require carefully orchestrated changes in cellular morphology. Single-celled eukaryotes provide tractable models for the elucidation of signaling pathways involved in morphogenesis. Here we describe a pathway regulating cell polarization and separation in the human pathogenic fungus Cryptococcus neoformans. An insertional mutagenesis screen identified roles for the ARF1, CAP60, NDH1, KIC1, CBK1, SOG2, and TAO3 genes in establishing normal colony morphology. ARF1 and CAP60 are also required for capsule production, a virulence factor, and ARF1 confers resistance to the antifungal fluconazole. KIC1, CBK1, SOG2, and TAO3 are homologues of genes conserved in other eukaryotes; in Saccharomyces cerevisiae they constitute components of the RAM (regulation of Ace2p activity and cellular morphogenesis) signaling pathway. A targeted deletion of a fifth component of RAM (MOB2) conferred identical phenotypes to kic1, cbk1, sog2, or tao3 mutations. Characterization of these genes in C. neoformans revealed unique features of the RAM pathway in this organism. Loss of any of these genes caused constitutive hyperpolarization instead of the loss of polarity seen in S. cerevisiae. Furthermore, sensitivity to the drugs FK506 and cyclosporin A demonstrates that the RAM pathway acts in parallel with the protein phosphatase calcineurin in C. neoformans but not in S. cerevisiae. These results indicate that conserved signaling pathways serve both similar and divergent cellular roles in morphogenesis in these divergent organisms.