Deciphering the model pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete fungal pathogen of humans that has diverged considerably from other model fungi such as Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae and the common human fungal pathogen Candida albicans. The recent completion of the genome sequences of two related C. neoformans strains and the ongoing genome sequencing of three other divergent Cryptococcus strains with different virulence phenotypes and environmental distributions should improve our understanding of this important pathogen. We discuss the biology of C. neoformans in light of this genomic data, with a special emphasis on the role that evolution and sexual reproduction have in the complex relationships of the fungus with the environment and the host.     

RNA interference in the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus responsible for serious disease in immunocompromised individuals. This organism has recently been developed as an experimental system, with initiation of a genome project among other molecular advances. However, investigations of Cryptococcus are hampered by the technical difficulty of specific gene replacements. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, may help solve this problem. We have shown that expression of double-stranded RNA corresponding to portions of the cryptococcal CAP59 and ADE2 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Specific modulation of protein expression through introduction of double-stranded RNA thus operates in C. neoformans, which is the first demonstration of this technique in a fungal organism. Use of RNA interference in Cryptococcus should allow manipulation of mRNA levels for functional analysis of genes of interest and enable efficient exploration of genes discovered by genome sequencing.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

A unique alpha-1,3 mannosyltransferase of the pathogenic fungus Cryptococcus neoformans.

The major virulence factor of the pathogenic fungus Cryptococcus neoformans is an extensive polysaccharide capsule which surrounds the cell. Almost 90% of the capsule is composed of a partially acetylated linear alpha-1,3-linked mannan substituted with D-xylose and D-glucuronic acid. A novel mannosyltransferase with specificity appropriate for a role in the synthesis of this glucuronoxylomannan is active in cryptococcal membranes. This membrane-associated activity transfers mannose in vitro from GDP-mannose to an alpha-1, 3-dimannoside acceptor, forming a second alpha-1,3 linkage. Product formation by the transferase is dependent on protein, time, temperature, divalent cations, and each substrate. It is not affected by amphomycin or tunicamycin but is inhibited by GDP and mannose-1-phosphate. The described activity is not detectable in the model yeast Saccharomyces cerevisiae, consistent with the absence of a similar polysaccharide structure in that organism. A second mannosyltransferase from C. neoformans membranes adds mannose in alpha-1,2 linkage to the same dimannoside acceptor. The two activities differ in pH optimum and cation preference. While the alpha-1,2 transferase does not have specificity appropriate for a role in glucuronoxylomannan synthesis, it may participate in production of mannoprotein components of the capsule. This study suggests two new targets for antifungal drug discovery.     

The origin and maintenance of virulence for the human pathogenic fungus Cryptococcus neoformans

The origin of virulence in environmental fungi that have no requirement for animal hosts in their life cycle is enigmatic. Cryptococcus neoformans is a human pathogenic fungus with virulence factors for mammalian pathogenesis that also contribute to environmental survival. C. neoformans virulence may originate from selection pressures imposed by environmental predators.     

UDP-glucose dehydrogenase plays multiple roles in the biology of the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals. Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites. To examine the role of these nucleotide sugars in C. neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted. Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used. The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects. Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display. The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology. All of the observed alterations were reversed by correction of the gene disruption.     

Cloning of 18S and 25S rDNAs from the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is an important pathogenic fungus that has been classified as a basidiomycete. Little is known of the molecular genetics of this fungal pathogen. To begin such studies, we devised a procedure for extraction of DNA from cryptococci; this method involved the use of the cell wall-active enzyme NovoZym 234. Using cloned rDNA of Saccharomyces cerevisiae as a probe, we identified homologous restriction fragments in a Southern blot of digested C. neoformans DNA. An 8.6-kilobase HindIII fragment that hybridized with the yeast rDNA probe was ligated with the vector pBR322 and cloned into Escherichia coli. When the fragment was used as a probe, it hybridized to the 18S and 25S rRNAs of C. neoformans in Northern (RNA) blots of native and denatured RNA. It bound at high stringency only weakly to the rRNAs of the ascomycete S. cerevisiae. The locations of the genes for 5/5.8S, 18S, and 25S subunits in the cloned fragment were identified with labeled rRNA of these different types.     

Genotype-environment interactions of spontaneous mutations for vegetative fitness in the human pathogenic fungus Cryptococcus neoformans

Spontaneous mutation is the ultimate source of all genetic variation. By interacting with environmental factors, genetic variation determines the phenotype and fitness of individuals in natural populations. However, except in a few model organisms, relatively little is known about the patterns of genotype-environment interactions of spontaneous mutations. Here I examine the rates of spontaneous mutation and the patterns of genotype-environment interaction of mutations affecting vegetative growth in the human fungal pathogen Cryptococcus neoformans. Eight mutation accumulation (MA) lines were established from a single clone on the nutrient-rich medium YEPD for each of two temperatures, 25 degrees and 37 degrees. Cells from generations 100, 200, 400, and 600 for each of the 16 MA lines were stored and assayed for vegetative growth rates under each of four conditions: (i) 25 degrees on SD (a synthetic dextrose minimal medium); (ii) 25 degrees on YEPD; (iii) 37 degrees on SD; and (iv) 37 degrees on YEPD. Both MA conditions and assay environments for vegetative growth showed significant influence on the estimates of genomic mutation rates, average effect per mutation, and mutational heritability. Significant genotype-environment interactions were detected among the newly accumulated spontaneous mutations. Overall, clones from MA lines maintained at 37 degrees showed less decline in vegetative fitness than those maintained at 25 degrees. The result suggests that a high-temperature environment might be very important for the maintenance of the ability to grow at a high temperature. Results from comparisons between clinical and environmental samples of C. neoformans were consistent with laboratory experimental population analyses. This study calls into question our long-standing view that warm-blooded mammals were only occasional and accidental hosts of this human fungal pathogen.     

Cryptococcus neoformans, a fungus under stress

Cryptococcus neoformans is a human fungal pathogen that survives exposure to stresses during growth in the human host, including oxidative and nitrosative stress, high temperature, hypoxia, and nutrient deprivation. There have been many genes implicated in resistance to individual stresses. Notably, the catalases do not have the expected role in resistance to external oxidative stress, but specific peroxidases appear to be critical for resistance to both oxidative and nitrosative stresses. Signal transduction through the HOG1 and calcineurin/calmodulin pathways has been implicated in the stress response. Microarray and proteomic analyses have indicated that the common responses to stress are induction of metabolic and oxidative stress genes, and repression of genes encoding translational machinery.     

Electrophoretic karyotype of the pathogenic yeast Cryptococcus neoformans

The electrokaryotype of the pathogenic yeast Cryptococcus neoformans is described for the first time. Three different patterns were seen: (a) serotypes B and C (variety gattii) are similar and consist of nine chromosome mobility groups of greater than 580 kb; (b) serotype A (variety neoformans) revealed eight chromosome-like groups greater than 700 kb; (c) serotype D (the second serotype of variety neoformans) not only differs from those described above, but each D isolate tested showed a different distribution of bands. The discrepancy, and the importance of electrokaryotyping as a taxonomic tool, are discussed.     

Estimating the spontaneous mutation rate of loss of sex in the human pathogenic fungus Cryptococcus neoformans

Few events have evolutionary consequences as pervasive as changes in reproductive behavior. Among those changes, the loss of the ability to undergo sexual reproduction is probably the most profound. However, little is known about the rate of loss of sex. Here I describe an experimental system using the fungus Cryptococcus neoformans and provide the first empirical estimate of the spontaneous mutation rate of loss of sex in fungi. Two critical steps in sexual reproduction in C. neoformans were examined: mating and filamentation. Mating, the fusion of cells of opposite sexes, is a universal first step in eukaryotic sexual reproduction. In contrast, filamentation, a prerequisite process preceding meiosis and sexual spore development, is restricted to C. neoformans and a few other fungal species. After approximately 600 mitotic divisions under favorable asexual growth conditions, mean abilities for mating and filamentation decreased significantly by >67 and 24%, respectively. Similarly, though statistically not significant, the mean vegetative growth rates also decreased and among the mutation accumulation lines, the vegetative growth rates were negatively correlated to the mating ability. The estimated mutation rates to decreases in mating ability and filamentation were in excess of 0.0172 and 0.0036, respectively. The results show that C. neoformans can be a highly attractive model for analyses of reproductive system evolution in fungi.     

Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa

Most of the species of fungi that cause disease in mammals, including Cryptococcus neoformans var. grubii (serotype A), are exogenous and non-contagious. Cryptococcus neoformans var. grubii is associated worldwide with avian and arboreal habitats. This airborne, opportunistic pathogen is profoundly neurotropic and the leading cause of fungal meningitis. Patients with HIV/AIDS have been ravaged by cryptococcosis--an estimated one million new cases occur each year, and mortality approaches 50%. Using phylogenetic and population genetic analyses, we present evidence that C. neoformans var. grubii may have evolved from a diverse population in southern Africa. Our ecological studies support the hypothesis that a few of these strains acquired a new environmental reservoir, the excreta of feral pigeons (Columba livia), and were globally dispersed by the migration of birds and humans. This investigation also discovered a novel arboreal reservoir for highly diverse strains of C. neoformans var. grubii that are restricted to southern Africa, the mopane tree (Colophospermum mopane). This finding may have significant public health implications because these primal strains have optimal potential for evolution and because mopane trees contribute to the local economy as a source of timber, folkloric remedies and the edible mopane worm.     

Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an 2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-¹⁴C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat 2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003.     

Characterization of pathogenic constituents of Cryptococcus neoformans strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 X 10(5) or 1 X 10(7) viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; a cell surface structure for responsible for resistance to intra- or extracellular killing and induction of the granulomatous lesion; a growth rate suitable for interacting with phagocytic elimination.     

Major histocompatibility complex controls the trajectory but not host-specific adaptation during virulence evolution of the pathogenic fungus Cryptococcus neoformans

Genes of the major histocompatibility complex (MHC) play a critical role in immune recognition and are the most genetically diverse loci known. One hypothesis to explain this diversity postulates that pathogens adapt to common MHC haplotypes and thus favour selection of new or rare alleles. To determine whether the pathogenic yeast Cryptococcus neoformans adapts to MHC-dependent immune responses, it was serially passaged in two independent replicate lines of five B10 MHC-congenic strains and Balb/c mice. All passaged lines increased in virulence as measured by reduced host survival. MHC influenced the rate (trajectory) of virulence increase during passages as measured by significant differences in mortality rate (p < 0.001). However, when the post-passage strains were tested, no MHC differences in mortality rate remained and only minor differences in titres were observed. Also contrary to expectations, increased virulence in three lines passaged in B10 mice had a larger effect in Balb/c mice, and the evolution of virulence in lines passaged in alternating hosts was not retarded. To our knowledge, these data represent the first experimental test of MHC-specific adaptation in a non-viral pathogen. The failure to observe MHC effects despite dramatically increased virulence and host-genotype-specific adaptation to non-MHC genes suggests that escape of MHC-dependent immune recognition may be difficult for pathogens with unlimited epitopes or that other virulence factors can swamp MHC effects.     

A Rac homolog functions downstream of Ras1 to control hyphal differentiation and high-temperature growth in the pathogenic fungus Cryptococcus neoformans

The Cryptococcus neoformans Ras1 protein serves as a central regulator for several signaling pathways. Ras1 controls the induction of the mating pheromone response cascade as well as a distinct signaling pathway that allows this pathogenic fungus to grow at human physiological temperature. To characterize elements of the Ras1-dependent high-temperature growth pathway, we performed a multicopy suppressor screen, identifying genes whose overexpression allows the ras1 mutant to grow at 37 degrees C. Using this genetic technique, we identified a C. neoformans gene encoding a Rac homolog that suppresses multiple ras1 mutant phenotypes. Deletion of the RAC1 gene does not affect high-temperature growth. However, a rac1 mutant strain demonstrates a profound defect in haploid filamentation as well as attenuated mating. In a yeast two-hybrid assay, Rac1 physically interacts with the PAK kinase Ste20, which similarly regulates hyphal formation in this fungus. Similar to Rac1, overexpression of the STE20alpha gene also restores high-temperature growth to the ras1 mutant. These results support a model in which the small G protein Rac1 acts downstream of Ras proteins and coordinately with Ste20 to control high-temperature growth and cellular differentiation in this human fungal pathogen.     

Genetic analyses of a hybrid cross between serotypes A and D strains of the human pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans has two varieties, var. grubii and var. neoformans, that correspond to serotypes A and D, respectively. Molecular phylogenetic analyses suggest that these two varieties have diverged from each other for approximately 18 million years. The discovery of pathogenic serotype AD hybrid strains in nature indicates that intervariety mating in C. neoformans occurs in the natural environment. However, little is known about the genetic consequences of hybridization in C. neoformans. Here, we analyzed a hybrid population of 163 progeny from a cross between strains of serotypes A (CDC15) and D (JEC20), using 114 codominant nuclear PCR-RFLP markers and 1 direct PCR marker. These markers were distributed on all 14 chromosomes of the sequenced strain JEC21 that was isogenic to one of the parents (JEC20) in our cross. Our analyses identified that of the 163 progeny, 5 were heterozygous at all 115 loci, 1 was completely homozygous and identical to one of the parents (CDC15), and the remaining 157 each contained at least 1 heterozygous locus. Because all 163 progeny inherited mitochondria from the MATa parent JEC20, none of the progeny had a genotype identical to either of the two parents or to a composite of the two parents. All 115 nuclear loci showed three different genotypes in the progeny population, consistent with Mendelian segregation during meiosis. While the linkage analysis showed independent reassortment among loci on different linkage groups, there were significant differences in recombination frequencies among chromosomes and among regions within certain chromosomes. Overall, the linkage-map length from this hybrid cross was much shorter and the recombination frequency much lower than those constructed using serotype D strains, consistent with suppressed recombination in the intervariety cross between strains of serotypes A and D. We discuss the implications of our results in our understanding of the speciation and evolution of the C. neoformans species complex.     

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.     

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.