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PubMed1999

Molecular cloning of homologs of RAS and RHO1 genes from Cryptococcus neoformans.

We cloned and sequenced homologs of RAS(CnRAS) and RHO1(CnRHO1) genes from Cryptococcus neoformans. The proteins encoded by the CnRAS and CnRHO1 genes contained 216 and 197 amino acids, respectively. The deduced amino acid sequence of the CnRAS gene shared a high degree of sequence identity with the Ras proteins in other fungal species: Coprinus cinereus(76%), Lentinula edodes(74%), Saccharomyces cerevisiae RAS2(72%), and Schizosaccharomyces pombe(68%). The deduced amino acid sequence of the CnRHO1 gene shared a high degree of sequence identity with the Rho1 proteins in other fungal species: Candida albicans(78%), S. pombe(77%) and S. cerevisiae(76%). The deduced proteins contained GTP-binding and GTP-hydrolysis domains, and the prenylation site that are conserved among the small G protein superfamily. The synthetic peptides that contained the C-terminal amino acid sequence of the CnRas and CnRho1 proteins were geranylgeranylated.

Amino Acid Sequence↗
PubMed1999

Inositol synthesis and catabolism in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that synthesizes and catabolizes inositol. This study demonstrates inositol synthesis from glucose-6-phosphate via inositol-1-phosphate synthase and catabolism to glucuronic acid via inositol oxygenase in this organism. These inositol synthetic and catabolic pathways are regulated in opposition; repressing conditions for one are inducing conditions for the other. An inositol-requiring strain was generated by UV mutagenesis. Without inositol, this mutant strain undergoes 'inositol-less' death, during which time the phosphatidylinositol composition of the membranes decreases without alteration of the proportion of other phospholipids. The mutation on this strain results in no detectable inositol synthetic activity but normal (wild-type) inositol catabolic activity. This inositol-requiring mutant strain reverted at a high frequency. Classical genetic experiments revealed that the majority of the reverting mutations are at second sites. Interestingly, the revertants exhibited unusual morphological phenotypes when deprived of inositol, while provision of inositol restored wild-type morphology. Inositol metabolism is clearly important for growth and development of C. neoformans and may be involved in this organism's mechanism for survival as both a saprophyte in soil and a parasite in humans.

Base Sequence↗
PubMed1998

Cryptococcus neoformans differently regulates B7-1 (CD80) and B7-2 (CD86) expression on human monocytes.

To induce a specific response in primary resting T cells, two signals must be provided by antigen-presenting cells (APC). The first antigen-specific signal is mediated by formation of the T cell receptor major histocompatibility complex molecule ternary complexes. The second signal is delivered by interaction of either B7-1 or B7-2 expressed by APC with CD28 or CTLA-4 on T cells. In this study, we examined the modulation of B7-1 and B7-2 molecules on human monocytes exposed to encapsulated or acapsular Cryptococcus neoformans or Candida albicans. In our experimental system, C. albicans or acapsular C. neoformans are able to induce B7-1 expression while the encapsulated yeast is a poor stimulator. A modest increase of B7-2 expression was also observed after monocyte treatment with acapsular C. neoformans or C. albicans, while the encapsulated yeast was ineffective in inducing B7-2 molecules. Kinetic analysis showed the maximum expression of B7-1 after 24 to 48 h. Addition of the opsonic IgG1 mAb 2H1 to monocytes and C. neoformans significantly increased B7-1, but not B7-2, expression. The contribution of B7-1 and B7-2 co-stimulatory (CS) molecules to cryptococcal-specific T cell activation was analyzed and a substantial inhibition of T cell proliferation was observed. In this study we provide the first demonstration of fungal interference in the regulation of CS molecules. Our results suggest a potential mechanism for poor inflammatory responses observed in C. neoformans infections.

Antigen Presentation↗
PubMed1999

Combined effects of IL-12 and IL-18 on the clinical course and local cytokine production in murine pulmonary infection with Cryptococcus neoformans.

We reported recently that interleukin (IL)-12 and IL-18 synergistically increased the fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by inducing the production of interferon (IFN)-gamma by natural killer (NK) cells. To confirm these findings in vivo, we examined the effect of combined treatment using these two cytokines on the course of experimentally induced pulmonary and disseminated cryptococcosis in mice. IL-12 and IL-18 were used at subtherapeutic doses (0.005 and 2 microg/mouse/day, respectively). A single administration of either cytokine was not effective in protecting mice against the infection, while combined treatment significantly prolonged survival time of infected mice and reduced the lung and brain loads of organisms. These protective effects were associated with elevated IFN-gamma and reduced IL-4 levels in bronchoalveolar lavage fluid. Finally, depletion of NK and gammadelta T cells, but not of CD4+ T cells, by administration of specific antibodies, significantly reduced the production of IFN-gamma in lungs by IL-12/IL-18 treatment during the 7 days of infection. Our results demonstrated that IL-12 and IL-18 protected mice against cryptococcal infection in a synergistic manner by enhancing the local production of IFN-gamma by NK and gammadelta T cells in the early phase of infection and by suppressing the production of IL-4 in lungs.

Animals↗
PubMed2000

T lymphocyte and monocyte interaction by CD40/CD40 ligand facilitates a lymphoproliferative response and killing of Cryptococcus neoformans in vitro.

This study explored the role of CD40 / CD40 ligand (CD40L) in the induction of a lymphoproliferative response and killing of Cryptococcus neoformans in vitro. In our experimental system, monocytes exposed to C. neoformans were used as antigen-presenting cells (APC) and co-cultured with autologous T cells. The results showed that CD40 / CD40L strongly regulated the blastogenic response of T cells to C. neoformans. The fungus up-regulated CD40 expression on APC. An acapsular strain appeared to be a better inducer than an encapsulated strain. Time course experiments showed optimal regulation of CD40 expression at 48 h of incubation. Blocking the interaction of CD40 on APC with CD40L on T cells using mAb to CD40L resulted in a significant inhibition of IFN-gamma production. The anti-cryptococcal activity of monocytes was greatly influenced by the CD40 / CD40L interaction, and a positive correlation was found between nitric oxide secretion and enhanced killing of C. neoformans. Finally, the CD40 / CD40L interaction was critical for induction of optimal secretion of pro-inflammatory cytokines such as TNF-alpha and IL-1beta. These results indicate an important role for CD40 / CD40L interaction in inducing activation of T cells. Such cell-to-cell contact promotes anti-cryptococcal activity as well as secretion of pro-inflammatory cytokines by monocytes.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1999

Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on which to base a detailed epidemiological survey.

A total of 356 clinical isolates of the encapsulated basidiomycetous fungus Cryptococcus neoformans var. neoformans, obtained from Australia, Argentina, Brazil, India, Italy, New Zealand, Papua New Guinea, South Africa, Thailand and the USA, were analyzed to lay the basis for a comprehensive evaluation of the global genetic structure of C. neoformans. Two polymerase chain reaction (PCR)-based typing techniques were standardized: PCR fingerprinting using a single primer specific to minisatellite or microsatellite DNA, and randomly amplified polymorphic DNA (RAPD) analysis using two combinations of three 20- to 22-mer random primers. Previous studies showed that the resultant profiles are reproducible and stable over time. Identical results were obtained in two different laboratories and by different scientists in the same laboratory. Both typing techniques separated the isolates into four major groups (VNI and VNII, serotype A; VNIII, serotype A/D; and VNIV, serotype D). The majority (78%) of isolates belonged to VNI, compared with 18% VNII, 1% VNIII and 3% VNIV. All US isolates could be differentiated by a unique, strain-specific PCR fingerprint or RAPD pattern in contrast to most of the non-US isolates, which showed a substantially higher degree of genetic homogeneity, with some clonality, in different parts of the world. Isolates obtained from the same patient at different times and from different body sites, had identical banding patterns. Both typing techniques should provide powerful tools for epidemiological studies of medically important fungi.

Base Sequence↗
PubMed2000

Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance.

A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild-type strain of C. neoformans which is sensitive to cycloheximide. The open reading frame of the L41 gene contains five introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild-type L41 gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10-20 microg/ml cycloheximide, at a maximum efficiency of 300 transformants/microg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformans maintain the transforming DNA as episomes, the cycloheximide-resistant transformants were all the result of ectopic genomic integration events.

Amino Acid Sequence↗
PubMed2002

Intra-strain variability of Cryptococcus neoformans can be detected on phloxin B medium.

A method was devised for easy detection of intra-strain variability of the human pathogenic yeast Cryptococcus neoformans. Cultivation of strains on a medium containing Phloxin B resulted in different coloured colonies. Generally, colonies were either pink or red; however there were also several colony-colour segregant in which both colours could be observed. A number of these segregants were isolated and analysed. Virulence factors such as the cell and capsule sizes were measured; further temperature sensitivity, growth rates, mating-types and melanin production were also studied. Segregants were examined by random amplified polymorphic DNA (RAPD) fingerprinting and electrophoretic karyotyping by pulsed-field gel electrophoresis (CHEF). They showed both phenotypic and genotypic differences. The main differences appeared in phenotypic characters and RAPD patterns; while the chromosomal patterns remained unchanged. Reversion frequency analysis revealed that the reason for this segregation could be due to phenotypic switching. The physiological reason for the colour changes was also investigated and was attributed to the differential ability of the cells to accumulate Phloxin B either into their capsules or into their cells. The method described here is potentially applicable for the detection of strain heterogeneity in both basic and clinical microbiology laboratories.

Cryptococcus neoformans↗
PubMed1992

Cytokine enhancement of complement-dependent phagocytosis by macrophages: synergy of tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor for phagocytosis of Cryptococcus neoformans.

We have examined the regulation of complement dependent phagocytosis by macrophage-activating cytokines. Tumor necrosis factor (TNF)-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF), but not interferon-gamma, interleukin-4 or macrophage-CSF, stimulated ingestion of the encapsulated fungal pathogen Cryptococcus neoformans by resident peritoneal macrophages in vitro. This was dependent upon opsonization of the yeasts with complement, 72 h of incubation with the cytokines for maximum effect, and the obligate involvement of the macrophage CR3 receptor. TNF-alpha and GM-CSF synergized at low concentrations, resulting in dramatic up-regulation of phagocytosis when compared to either cytokine alone. Supernatants from C. neoformans-specific T cells also increased macrophage phagocytic efficiency. Finally, the administration of neutralizing mAb specific for TNF-alpha and GM-CSF increased mortality in C. neoformans-infected mice, and induced the rapid progression of disease with involvement of the brain and meninges. We conclude that TNF-alpha and GM-CSF are potent regulators of complement-dependent phagocytosis by murine macrophages. Macrophage activation with these two cytokines can completely overcome the anti-phagocytic properties of the virulent yeasts. Our results, therefore, implicate TNF-alpha and GM-CSF as important mediators of resistance to encapsulated pathogens such as C. neoformans where ingestion of the organism is a critical process in host resistance.

Animals↗
PubMed2003

Differences in outcome of the interaction between Cryptococcus neoformans glucuronoxylomannan and human monocytes and neutrophils.

Disseminated infections by the opportunistic yeast Cryptococcus neoformans are characterized by accumulation in tissues of glucuronoxylomannan (GXM), the major component of the capsular polysaccharide. We investigated binding, uptake, and disposal of GXM by peripheral blood neutrophils and monocytes, and the effect of GXM uptake on phagocytic cell function. GXM was efficiently bound and internalized by both types of phagocytic cells, with maximal loading at 50 microg/ml, a GXM concentration found in serum and cerebrospinal fluid of some cryptococcosis patients. However, substantial differences were noted in the kinetics for uptake by macrophages and neutrophils. Whereas neutrophils rapidly ingested limited amounts of GXM and then expelled or degraded it after 1 h of incubation, macrophages demonstrated continuous intracellular accumulation for up to 1 week of incubation. Accumulation of GXM by neutrophils was accompanied by reduced anticryptococcal activity, suggesting one more mechanism for virulence enhancement by the major capsular component of C. neoformans.

Antigens, Fungal↗
PubMed2003

The efficacy of complement-mediated phagocytosis of Cryptococcus neoformans is dependent on the location of C3 in the polysaccharide capsule and involves both direct and indirect C3-mediated interactions.

Complement component 3 (C3) is the major opsonin for the pathogenic fungus Cryptococcus neoformans in the non-immune host. However, the efficiency of complement-mediated opsonization varies, depending on the strain, through mechanisms that are not understood. Analysis of complement-mediated phagocytosis for 12 strains grown in Sabouraud medium revealed that phagocytic indices were inversely correlated with capsule volume. In contrast, there was no correlation between phagocytic index and capsule volume for IgG1-opsonized cells. When capsule size was increased, the efficacy of complement-mediated phagocytosis decreased, whereas that of antibody-mediated phagocytosis increased. C3 localized inside the capsule and at the outer capsule edge for poorly phagocytozed and well-phagocytozed strains, respectively. Blocking experiments revealed that complement-mediated phagocytosis occurred through complement receptor 3 (CR3), without significant involvement of CR1 or CR4. Blocking experiments with antibodies to C3 did not completely abrogate yeast cell uptake, consistent with phagocytosis through glucuronoxylomannan-CR3 interactions. Our data explain how some large encapsulated cells avoid phagocytosis and suggest a novel strategy for immune evasion whereby a microbial capsule interferes with phagocytosis by modifying the location of C3 deposition.

Complement C3↗
PubMed1995

Polymerase chain reaction fingerprinting in fungi using single primers specific to minisatellites and simple repetitive DNA sequences: strain variation in Cryptococcus neoformans.

Minisatellites and simple repetitive DNA sequence motifs are used as conventional oligonucleotide probes in DNA-hybridization-based fingerprinting. The same oligonucleotides can be used as single primers in the polymerase chain reaction (PCR) to generate individual PCR fingerprints. In this study, the simple repetitive sequences, (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, and a minisatellite core sequence derived from the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3') were used as specific, single primers to amplify hypervariable repetitive DNA sequences during PCR analysis. The potential applications of this techniques are demonstrated with clinical isolates of the human pathogenic yeast, Cryptococcus neoformans. PCR fingerprint patterns have remained stable after long-term in vitro passage ( > 2 1/2 years to date). Hybridization of the primers to blots of electrophorectically separated chromosomes demonstrated that the target sequences recognized by most of the primers are dispersed through the entire yeast genome. Sequence analysis of the cloned bands obtained by PCR fingerprinting indicated that if the same or extremely similar, inversely oriented tandem repeats are located close to each other, when only one repeat-specific primer is used in the PCR, the region between these repeats is amplified. PCR fingerprinting has a wide range of current and potential applications to fungi, such as clarifying taxonomic questions, facilitating epidemiological studies and improving the diagnosis of mycotic diseases.

Base Sequence↗
PubMed2002

MHC class II-positive perivascular microglial cells mediate resistance to Cryptococcus neoformans brain infection.

Acquired resistance to the CNS pathogen Cryptococcus neoformans is mediated by CD4(+) T lymphocytes primed by exposure to antigen in the context of major histocompatibility class II (MHC II) molecules. In mouse brain, parenchymal and perivascular microglial cells may express interferon-gamma (IFN-gamma)-inducible MHC class II marker and thus interact with CD4(+) T cells. Primed effector T cells are retained in the infected CNS if antigen is encountered in proper MHC context and may deliver signals that potentiate microglia to enhanced fungistasis. Vaccinated C57BL6/J mice resist an ordinarily lethal C. neoformans rechallenge, but identically treated congenic Abeta(o/o) mice (MHC class II-deficient; CD4(+) T-cell-deficient) do not. Nor can Abeta(o/o) mice be adoptively immunized by infusion of lymphocytes from vaccinated C57BL6/J donors, as are severe combined immunodeficient (SCID) mice (MHC class II-intact, lymphocyte-deficient). Chimeric (C57BL/6J:Abeta(o/o)) mice with class II expression likely on perivascular microglia only were, like SCID mice, capable of adoptive immunization against C. neoformans brain infection. To the contrary, chimeric mice with class II expression likely only on parenchymal microglia were not capable of effective adoptive immunization against C. neoformans brain infection. Therefore, in order to mediate resistance to infection, primed CD4(+) T cells must interact with the replenishable perivascular microglial subset that lies in close proximity to cerebral vasculature. Although T cells may supply help in the form of inflammatory cytokines to parenchymal microglia, expression of class II on these cells appears unnecessary for antifungal activity.

Animals↗
PubMed2001

The diversity of retrotransposons in the yeast Cryptococcus neoformans.

We have undertaken an analysis of the retrotransposons in the medically important basidiomycetous fungus Cryptococcus neoformans. Using the data generated by a C. neoformans genome sequencing project at the Stanford Genome Technology Center, 15 distinct families of LTR retrotransposons and several families of non-LTR retrotransposons were identified. Members of at least seven families have transposed recently and are probably still active. For several families, only partial elements could be identified and these are quite diverse in sequence, suggesting that they are ancient components of the C. neoformans genome. Most C. neoformans elements are not closely related to previously identified fungal retrotransposons, suggesting that the diversity of fungal retrotransposons has been only sparsely sampled to date. C. neoformans has fewer distinct retrotransposon families than Candida albicans (37 or more), in particular fewer families represented solely by ancient and inactive elements, but it has considerably more families than either Saccharomyces cerevisiae (five) or Schizosaccharomyces pombe (two). The findings suggest that elimination of retrotransposons is faster in C. neoformans than in C. albicans, but perhaps not as rapid as in S. cerevisiae or Sz. pombe. The identification of the retrotransposons of C. neoformans should assist in the molecular characterization of this important pathogen, and also further our understanding of the role played by retroelements in genome evolution.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of glucose-6-phosphate dehydrogenase from Cryptococcus neoformans: identification as "nothing dehydrogenase".

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from Cryptococcus neoformans, a basidiomyceteous yeast that is an opportunistic pathogen of AIDS patients. The enzyme had a subunit molecular weight of 5 x 10(4), a specific activity of 50 units mg-1, and Km values for NADP and glucose-6-phosphate of 1.6 and 24 microM, respectively. The enzyme catalyzed the dehydrogenation of glucose, in the presence of dimethylsulfoxide, with Km of 5 mM and Vmax 10% of that with glucose-6-phosphate. pH profiles indicated the presence of a group with pKa of 6.6 that is involved in catalysis, and groups with pKas of about 8.8 that are involved in binding of NADP and glucose-6-phosphate. The enzyme was inhibited by NADPH, competitive versus NADP, with Ki of 1 microM, and by zinc ion, competitive versus glucose-6-phosphate, with Ki of 2 microM. Crude enzyme extract catalyzed an appreciable rate of reduction of NADP in the absence of added substrate, a "nothing dehydrogenase" activity. This activity was shown to be due to the presence of glucose-6-phosphate in the crude extract. It was calculated that cells of C. neoformans contain about 25 mumol of glucose-6-phosphate per gram, wet weight.

Chromatography, Affinity↗
PubMed1995

Polyethylene sulfonate: a tight-binding inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans.

Polyethylene sulfonate (PES) or polyvinyl sulfonate was found to be a potent inhibitor of a number of fungal enzymes, including 6-phosphogluconate dehydrogenase from Cryptococcus neoformans. The inhibition was apparently competitive versus either NADP or 6-phosphogluconate, with 50% inhibition at PES concentrations below 10 nM. Replots of slopes of double-reciprocal plots versus inhibitor concentration were sharply concave upward, whereas replots of slope versus [PES]3 were linear. The inhibition was freely reversible upon dilution of the enzyme-PES complex. A model is presented that involves initial binding of the long (M(r) 50,000) polyanionic PES at a remote site on the enzyme, followed by interaction of the end of the tethered polymer with the binding site for NADP or for 6-phosphogluconate.

Alkanesulfonic Acids↗
PubMed1995

Purification and characterization of malate dehydrogenase from Cryptococcus neoformans.

The NAD-dependent malate dehydrogenase (EC 1.1.1.37) was purified from Cryptococcus neoformans, a basidiomycetious yeast that is an opportunistic pathogen of AIDS patients. The purified enzyme was a dimer of 35 kDa subunits that exhibited uncompetitive substrate inhibition by oxalacetate, typical for mitochondrial malate dehydrogenases from other sources. Product inhibition studies indicated an ordered sequential kinetic mechanism, with pyridine dinucleotide being the substrate that binds to the free enzyme form. Unique aspects of this malate dehydrogenase were inhibition by zinc ion, competitive versus malate with Ki of 30 microM, and inhibition by heparin. Heparin inhibition was competitive versus either NAD or malate, with Ki of 0.35 microM. Heparin molecules of nominal molecular weight of 30,000 or 3000 were equally effective inhibitors. A model is presented to explain the high affinity of the enzyme for heparin.

AIDS-Related Opportunistic Infections↗
PubMed1998

Biochemical role of the Cryptococcus neoformans ADE2 protein in fungal de novo purine biosynthesis.

Comparative studies of 5-aminoimidazole ribonucleotide (AIR) carboxylases from Escherichia coli and Gallus gallus have identified this central step in de novo purine biosynthesis as a case for unusual divergence in primary metabolism. Recent discoveries establish the fungal AIR carboxylase, encoded by the ADE2 gene, as essential for virulence in certain pathogenic organisms. This investigation is a biochemical analysis that links the fungal ADE2 protein to the function of the E. coli AIR carboxylase system. A cDNA clone of ADE2 from Cryptococcus neoformans was isolated by genetic complementation of a purE-deficient strain of E. coli. High-level expression of the C. neoformans ADE2 was achieved, which enabled the production and purification of AIR carboxylase. Amino acid sequence alignments, C-terminal deletion mutants, and biochemical assays indicate that the ADE2 enzyme is a two-domain, bifunctional protein. The N-terminal domain is related to E. coli PurK and a series of kinetic experiments show that the ADE2-PurK activity uses AIR, ATP, and HCO3- as substrates. The biosynthetic product of the ADE2-PurK reaction was identified as N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) by 1H NMR, thus confirming that the C-terminal domain contains a catalytic activity similar to that of the E. coli PurE. By using an in situ system for substrate production, the steady-state kinetic constants for turnover of N5-CAIR by ADE2 were determined and together with stoichiometry measurements, these data indicate that ADE2 has a balance in the respective catalytic turnovers to ensure efficient flux. Distinctive features of the PurE active site were probed using 4-nitro-5-aminoimidazole ribonucleotide (NAIR), an analog of the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). NAIR was shown to be a selective inhibitor of the ADE2-PurE activity (K1 = 2.4 microM), whereas it is a slow-binding inhibitor of the G. gallus enzyme which further distinguishes the fungal ADE2 from the G. gallus AIR carboxylase. As such, this enzyme represents a novel intracellular target for the discovery of antifungal agents.

Adenosine Triphosphate↗