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.     

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.     

Mapping of the Cryptococcus neoformans MATalpha locus: presence of mating type-specific mitogen-activated protein kinase cascade homologs

In this study we investigated the relationship between the MATalpha locus of Cryptococcus neoformans and several MATalpha-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12alpha, STE11alpha, and STE20alpha. To resolve the location of the genes, we screened a cosmid library of the MATalpha strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MATalpha locus. It was found that STE12alpha, STE11alpha, and STE20alpha are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFalpha, the mating type alpha-pheromone gene, a MATalpha-specific myosin gene, and a pheromone receptor (CPRalpha) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATalpha locus. The MATalpha locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.     

The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle. The gene encoding a heterotrimeric G-protein beta subunit, GPB1, was cloned and disrupted. gpb1 mutant strains are sterile, indicating a role for this gene in mating. GPB1 plays an active role in mediating responses to pheromones in early mating steps (conjugation tube formation and cell fusion) and signals via a mitogen-activated protein (MAP) kinase cascade in both MATalpha and MATa cells. The functions of GPB1 are distinct from those of the Galpha protein GPA1, which functions in a nutrient-sensing cyclic AMP (cAMP) pathway required for mating, virulence factor induction, and virulence. gpb1 mutant strains are also defective in monokaryotic fruiting in response to nitrogen starvation. We show that MATa cells stimulate monokaryotic fruiting of MATalpha cells, possibly in response to mating pheromone, which may serve to disperse cells and spores to locate mating partners. In summary, the Gbeta subunit GPB1 and the Galpha subunit GPA1 function in distinct signaling pathways: one (GPB1) senses pheromones and regulates mating and haploid fruiting via a MAP kinase cascade, and the other (GPA1) senses nutrients and regulates mating, virulence factors, and pathogenicity via a cAMP cascade.     

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.     

Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic

Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATalpha and MATa strains. At 37 degrees C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATalpha and MATa loci. At 24 degrees C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.     

Haploid fruiting in Cryptococcus neoformans is not mating type alpha-specific

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATalpha strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATalpha-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.     

Blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in Cryptococcus neoformans

Cryptococcus neoformans is a heterothallic basidiomycetous yeast that primarily infects immunocompromised individuals. Dikaryotic hyphae resulting from the fusion of the MATa and MATalpha mating type strains represent the filamentous stage in the sexual life cycle of C. neoformans. In this study we demonstrate that the production of dikaryotic filaments is inhibited by blue light. To study blue light photoresponse in C. neoformans, we have identified and characterized two genes, CWC1 and CWC2, which are homologous to Neurospora crassa wc-1 and wc-2 genes. Conserved domain analyses indicate that the functions of Cwc1 and Cwc2 proteins may be evolutionally conserved. To dissect their roles in the light response, the CWC1 gene deletion mutants are created in both mating type strains. Mating filamentation in the bilateral cross of cwc1 MATa and MATalpha strains is not sensitive to light. The results indicate that Cwc1 may be an essential regulator of light responses in C. neoformans. Furthermore, overexpression of the CWC1 or CWC2 gene requires light activation to inhibit sexual filamentation, suggesting both genes may function together in the early step of blue light signalling. Taken together, our findings illustrate blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans.     

On the origins of congenic MATalpha and MATa strains of the pathogenic yeast Cryptococcus neoformans.

The basidiomycetous yeast Cryptococcus neoformans infects humans and causes a meningoencephalitis that is uniformly fatal if untreated. The organism has a defined sexual cycle involving mating of haploid MATa and MATalpha strains, gene disruption by transformation and homologous recombination is now readily accomplished, and robust animal models for infection have been well established. In addition, a pair of congenic MATalpha and MATa haploid strains have been constructed that permit detailed studies on physiology and virulence by classical genetic approaches. These strains represent a valuable resource for further studies in this organism, and the genomic sequence of one of these strains, JEC21 (=B-4500), was recently chosen to be sequenced by an international consortium. Because of the importance of these strains for genetic studies in C. neoformans and the fact that the genomic sequence of one of these strains is in progress, we review here how these congenic strains were originally constructed.     

Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.     

Environment factors can influence mitochondrial inheritance in the fungus Cryptococcus neoformans

Cryptococcus neoformans is a model basidiomycete yeast. Strains of this species belong to one of two mating types: mating type a (MATa) or mating type alpha (MATalpha). In typical crosses between MATa and MATalpha strains, the progeny inherit mitochondria from the MATa parent. However, the underlying mechanisms remain largely unknown. To help elucidate the molecular mechanisms, we examined the effects of four environmental factors on the patterns of mtDNA inheritance. These factors are temperature, UV irradiation, and the addition of either the methylation inhibitor 5-aza-2'-deoxycytidine (5-adc) or the ubiquitination inhibitor ammonium chloride. Except temperature, the other three factors have been shown to influence organelle inheritance during sexual mating in other eukaryotes. Our results indicate that while the application of 5-adc or ammonium chloride did not influence mtDNA inheritance in C. neoformans, both UV irradiation and high temperature treatments did. Progeny from a cross involving a high temperature-sensitive mutant with the calcineurin subunit A gene deleted showed biparental mtDNA inheritance in all examined temperatures, consistent with a role of calcineurin and temperature in mtDNA inheritance. Furthermore, the zygote progeny population from a cross performed at a high-temperature environment had a greater variability in their vegetative fitness than that from the same cross conducted at a low temperature. Our results indicate a potentially adaptive role of biparental mtDNA inheritance and mtDNA recombination in certain environments in C. neoformans.     

Characterization of novel peptide agonists of the alpha mating factor of Saccharomyces cerevisiae.

Alpha-factor [WHWLQLKPGQPMY], a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae (MATa, MATalpha). New analogues of alpha-factor were synthesized and evaluated by morphogenesis assays and receptor binding studies. The Y(0)Nle(12)F(13) analogue [YWHWLQLKPGQPNleF] (MFN5) caused growth arrest and morphological alteration in MATa cells in a fashion identical to that of the native pheromone. Binding of (125)I-labeled MFN5 was saturable, and reversible as shown by equipotent label displacement by MFN5 and native alpha-mating factor. Scatchard analysis of equilibrium binding data on plasma membranes and intact cells indicated the existence of a single high-affinity binding site (K(d) = 6.4 x 10(-8)). Specific binding of (125)I-labeled MFN5 was significantly reduced by guanosine nucleotides. Affinity cross-linking of (125)I-labeled MFN5 to MATa cell membranes identified a specifically labeled 49-kDa protein. The novel synthetic alpha-factor analogue MFN5 can be easily iodinated and used as a probe for the alpha-factor receptor.     

Roles of sexual cell agglutination in yeast mass mating

The agalpha1 mutant MAT alpha cells specifically lack the cell surface alpha-type sexual agglutination substance, which is also called alpha-agglutinin. Because the mutant cells (MATalpha agalpha1) can not form aggregates with MATa cells, MATalpha agalpha1 cells are unable to mate with MATa cells when they are co-inoculated in a liquid medium, and the mating is attenuated on solid medium. The attenuated mating ability shown in the previous studies gave us a vague idea about a physiological function of the sexual agglutinability. In order to solve the question, mating behavior of MATalpha agalpha1 cells was investigated here under conditions where the contact between MATa and MAT alpha cells is assisted by physical methods. A synthetic mutation agalpha1::URA3 was constructed and used as well as agalpha1-1 for this study to ensure the genetic defect. When a mixture of MATa and MAT alpha cells was kept on filter membrane placed on relatively dry agar medium, even agalpha1::URA3 mutant cells mated as efficiently as the wild type (AGalpha1) cells did. On filter membrane placed on moist agar medium, agalpha1 mutants mated 10-fold less efficiently than wild type cells did. The mutant cells mated 10000-time less efficiently than the wild type cells in a pellet formed by brief low speed centrifugation. In contrast, the wild type MATalpha cells mated well under all conditions tested. Under the pellet condition, a mixture of MATa and MATalpha AG alpha1 cells formed an extended and cotton-like pellet while a mixture of MATa and MATalpha agalpha1 cells formed a compact and tight pellet. These results suggest that sexual cell agglutination contributes not only to cell contact between MATa and MAT alpha cells thereby stabilizing a-alpha cell pairs, but also to construction of a uniquely organized ultra structure favorable for zygote formation and subsequent growth of diploid cells. The mating specific extended pellet formation was observed also in 4 pairs of a and alpha strains in ascosporogenous yeast genera Hansenula and Pichia.     

A CIS-Acting Mutation within the MATa Locus of SACCHAROMYCES CEREVISIAE That Prevents Efficient Homothallic Mating-Type Switching

Homothallic strains of Saccharomyces cerevisiae are able to switch from one mating-type to the other as frequently as every cell division. We have identified a cis-dominant mutation of the MATa locus, designated MATa-inc, that can be converted to MATalpha at only about 5% of the normal efficiency. In homothallic MATa-inc/mata* diploids, the MATa-inc locus switched to MATalpha in only one of 30 cases, while the mata* locus switched to MATalpha in all 30 cases. The MATa-inc mutation can be "healed" by a series of switches, first to MATalpha and then to a normal allele of MATa. These data are consistent with the "cassette" model of Hicks, Strathern and Herskowitz (1977), in which mating conversions involve the transposition of wild-type copies of a or alpha information from silent genes elsewhere in the genome. The MATa-inc mutation appears to alter a DNA sequence necessary for the replacement of MATa by MATalpha. The MATa-inc mutation has no other effect on MATa functions. In beterothallic backgrounds, the mutation has no effect on the sensitivity to alpha-factor, synthesis of a-factor, expression of barrier phenotype or ability to mate or sporulate.--The MATa-inc allele does, however, exhibit one pleiotropic effect. About 1% of homothallic MATa-inc cells become completely unable to switch mating type because of mutations at HMa, the locus proposed to carry the silent copy of alpha information.--In addition, we have isolated a less efficient allele of the HO gene.     

Mitochondria are inherited from the MATa parent in crosses of the basidiomycete fungus Cryptococcus neoformans

Previous studies demonstrated that mitochondrial DNA (mtDNA) was uniparentally transmitted in laboratory crosses of the pathogenic yeast Cryptococcus neoformans. To begin understanding the mechanisms, this study examined the potential role of the mating-type locus on mtDNA inheritance in C. neoformans. Using existing isogenic strains (JEC20 and JEC21) that differed only at the mating-type locus and a clinical strain (CDC46) that possessed a mitochondrial genotype different from JEC20 and JEC21, we constructed strains that differed only in mating type and mitochondrial genotype. These strains were then crossed to produce hyphae and sexual spores. Among the 206 single spores analyzed from six crosses, all but one inherited mtDNA from the MATa parents. Analyses of mating-type alleles and mtDNA genotypes of natural hybrids from clinical and natural samples were consistent with the hypothesis that mtDNA is inherited from the MATa parent in C. neoformans. To distinguish two potential mechanisms, we obtained a pair of isogenic strains with different mating-type alleles, mtDNA types, and auxotrophic markers. Diploid cells from mating between these two strains were selected and 29 independent colonies were genotyped. These cells did not go through the hyphal stage or the meiotic process. All 29 colonies contained mtDNA from the MATa parent. Because no filamentation, meiosis, or spore formation was involved in generating these diploid cells, our results suggest a selective elimination of mtDNA from the MATalpha parent soon after mating. To our knowledge, this is the first demonstration that mating type controls mtDNA inheritance in fungi.     

Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae.

The STE4 gene of Saccharomyces cerevisiae encodes the beta subunit of the yeast pheromone receptor-coupled G protein. Overexpression of the STE4 protein led to cell cycle arrest of haploid cells. This arrest was like the arrest mediated by mating pheromones in that it led to similar morphological changes in the arrested cells. The arrest occurred in haploid cells of either mating type but not in MATa/MAT alpha diploids, and it was suppressed by defects in genes such as STE12 that are needed for pheromone response. Overexpression of the STE4 gene product also suppressed the sterility of cells defective in the mating pheromone receptors encoded by the STE2 and STE3 genes. Cell cycle arrest mediated by STE4 overexpression was prevented in cells that either were overexpressing the SCG1 gene product (the alpha subunit of the G protein) or lacked the STE18 gene product (the gamma subunit of the G protein). This finding suggests that in yeast cells, the beta subunit is the limiting component of the active beta gamma element and that a proper balance in the levels of the G-protein subunits is critical to a normal mating pheromone response.