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.     

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.     

RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete yeast and opportunistic human pathogen of increasing clinical importance due to the increasing population of immunocompromised patients. To further investigate signal transduction cascades regulating fungal pathogenesis, we have identified the gene encoding a RAS homologue in this organism. The RAS1 gene was disrupted by transformation and homologous recombination. The resulting ras1 mutant strain was viable, but failed to grow at 37 degrees C, and exhibited significant defects in mating and agar adherence. The ras1 mutant strain was also avirulent in an animal model of cryptococcal meningitis. Reintroduction of the wild-type RAS1 gene complemented these ras1 mutant phenotypes and restored virulence in animals. A dominantly active RAS1 mutant allele, RAS1Q67L, induced a differentiation phenotype known as haploid fruiting, which involves filamentation, agar invasion and sporulation in response to nitrogen deprivation. The ras1 mutant mating defect was suppressed by overexpression of MAP kinase signalling elements and partially suppressed by exogenous cAMP. Additionally, cAMP also suppressed the agar adherence defect of the ras1 mutant. However, the ability of the ras1 mutant strain to grow at elevated temperature was not restored by cAMP or MAP kinase overexpression. Our findings support a model in which RAS1 signals in C. neoformans through cAMP-dependent, MAP kinase, and RAS-specific signalling cascades to regulate mating and filamentation, as well as growth at high temperature which is necessary for maintenance of infection.     

Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae

In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth. At least two signaling pathways regulate filamentation. One involves components of the MAP kinase cascade that also regulates mating of haploid cells. The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A. Recent studies reveal crosstalk between these pathways during pseudohyphal growth. Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.     

Melanin and virulence in Cryptococcus neoformans

Melanin synthesis has been associated with virulence for the human pathogenic fungus Cryptococcus neoformans. Recent evidence indicates that C. neoformans cells synthesize melanin during infection and that this pigment protects the fungus against immune defense mechanisms.     

Mechanisms of unisexual mating in Cryptococcus neoformans

Sex serves a pivotal role in genetic exchange and it contributes to the fitness and genetic diversity for eukaryotic populations. Although the importance of the canonical bisexual mating has been widely accepted, the significance of the evolution and maintenance of unisexual mating observed in some eukaryotes is unclear. The recent discovery of same-sex mating in the human fungal pathogen Cryptococcus neoformans and the revelation of its impact on the Cryptococcus global population structure provide a platform to elucidate the molecular mechanisms and significance of unisexual mating. Here, we review the evidence of unisexual mating in Cryptococcus and provide some perspective on the biological significance of this life style on the survival of this important fungal pathogen in the environment and in animal hosts. We also summarize our current understanding of the molecular mechanisms governing this unconventional mode of reproduction.     

Calcineurin is required for virulence of Cryptococcus neoformans.

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.     

Intersection of fungal fitness and virulence in Cryptococcus neoformans

The use of insertional mutagenesis to discover genes that impact laccase activity has resulted in the identification of multiple cellular processes that affect the fitness of Cryptococcus neoformans. Fitness has been defined as the ability of an organism to propagate and evolve within a given environment. Because the human host is an evolutionary dead-end for an opportunistic pathogen, we have defined pathogenic fitness here as the capability to successfully propagate within the stressful environment of the host, causing disease by expression of virulence traits that damage the host. In this review, laccase-deficient insertional mutants will be highlighted in terms of the basic biological processes in which they are involved. The impact of laccase-associated cellular functions on fitness and virulence will be discussed, as will the mutants' potential as therapeutic targets. Vacuolar function, copper homeostasis, mitochondrial function and carbon repression are covered.     

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.     

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.     

Signal transduction during mating and meiosis in S. pombe

When starved, the fission yeast Schizosaccharomyces pombe responds by producing mating factors or pheromones that signal to cells of the opposite sex to initiate mating. Like its distant relative Saccharomyces cerevisiae, cells of the two mating types of S. pombe each produce a distinct pheromone that binds to receptors on the opposite cell type to induce the morphological changes required for mating. While the pathways are basically very similar in the two yeasts, pheromone signalling in S. pombe differs in several important ways from that of the more familiar budding yeast. In this article, Olaf Nielsen describes the pheromones and their effects in S. pombe, and compares the signalling pathways of the two yeasts.     

Role of laccase in the biology and virulence of Cryptococcus neoformans

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.     

Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice

A double mutant of Cryptococcus neoformans which lacked the ability to produce melanin (Mel-) on media containing diphenols and failed to grow at 37 degrees C (temperature sensitive, Tem-) was obtained by UV irradiation and subsequent cloning. The mutant showed two lesions in melanogenesis in that it lacked the active transport system for diphenolic compounds and also lacked phenoloxidase. Ultrastructures of the mutant and wild-type cells grown on a medium with or without L-dopa showed that only the wild-type cells grown on L-dopa medium formed a dark cell wall layer, presumably containing melanin. The mutant was crossed with a wild type, and the phenotypes of the progeny were analyzed. The analysis showed no linkage between the mating type and either Mel or Tem loci, but loose linkage was seen between Mel and Tem loci. The progeny, Mel+ Tem+, Mel+ Tem-, Mel- Tem+, and Mel- Tem-, were studied for their virulence in mice. Only Mel+ Tem+ types killed mice with an inoculum of 5 X 10(5) cells within 50 days.     

Role of laccase in the biology and virulence of Cryptococcus neoformans

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.     

Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms

Cryptococcus neoformans is the causative agent of cryptococcosis and cryptococcal meningitis, which are serious pathological conditions affecting up to 10% of patients with AIDS. Mechanisms of pathogenicity of C. neoformans and the host defenses against this fungus are reviewed, incorporating recent data and perspectives.