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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↗
PubMed1997

Cryptococcus neoformans inhibits nitric oxide production by murine peritoneal macrophages stimulated with interferon-gamma and lipopolysaccharide.

We examined the effect of Cryptococcus neoformans on nitric oxide (NO) production by activated cultured macrophages. C. neoformans suppressed NO production by murine peritoneal macrophages stimulated with bacterial lipopolysaccharide (LPS) and interferon (IFN)-gamma, while it did not influence the production of IL-1 beta. This effect was observed when 1 x 10(6) or 10(7) of C. neoformans was added to macrophage cultures. A direct contact of C. neoformans with macrophages was essential for this inhibitory effect, since placement of a 0.45-micron-pore membrane between the organism and macrophages prevented such effect. In addition, C. neoformans killed by heat or paraformaldehyde did not show this inhibitory activity. Capsular polysaccharide did not mediate the inhibitory effect, since two nonencapsulated mutant strains of C. neoformans showed an inhibitory activity similar to that of encapsulated wild strains, and culture supernatants of C. neoformans, rich in polysaccharide antigens, did not inhibit macrophage NO production compared with control culture medium. The inhibitory effect was also not mediated by interleukin (IL)-10 and transforming growth factor (TGF)-beta since neutralizing specific antibodies to these cytokines did not influence C. neoformans-induced reductions in macrophage NO production. Our results suggest that C. neoformans may cause a direct suppression of NO-mediated fungicidal activity of macrophages, and the effect is independent of the capsular polysaccharide and production of IL-10 and TGF-beta.

Animals↗
PubMed1999

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18.

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.

Animals↗
PubMed1997

Phagocytosis of nonopsonized Cryptococcus neoformans by swine microglia involves CD14 receptors.

The interaction of the opportunistic fungus Cryptococcus neoformans with swine microglia was studied in vitro in the presence and absence of anti-CD14 monoclonal antibodies. In the absence of anti-CD14 antibodies, 36% of microglia had phagocytized nonopsonized, encapsulated cryptococci after 2 hr incubation (effector-to-target ratio, 1:50). Preincubation of microglia with anti-CD14 antibodies resulted in a 63% reduction of phagocytosis. These findings suggest that CD14 receptors facilitate uptake of nonopsonized C, neoformans by resident macrophages within the brain.

Animals↗
PubMed1998

Morphine enhances complement receptor-mediated phagocytosis of Cryptococcus neoformans by human microglia.

Recent studies have shown that opiates modulate the function of microglia, the resident macrophages of the brain. In this study, the effect of morphine on phagocytosis by human fetal microglial cells of the opportunistic fungus Cryptococcus neoformans was studied. Contrary to earlier findings with swine microglia, opsonization was required for the phagocytosis of C. neoformans by human microglia. Moreover, morphine (10(-8)M) was shown to augment uptake of opsonized C. neoformans by over 50%. This contrasts with the earlier finding of morphine-induced inhibition of phagocytosis of nonopsonized cryptococci by swine microglia. The effect of morphine on cryptococcal phagocytosis by human microglia was reversed by treatment of microglial cells with mu opiate receptor antagonists as well as by addition of anti-complement receptor antibodies to the cell cultures, indicating that both the mu opiate receptor and the complement receptor are involved in morphine-enhanced phagocytosis. These findings support the concept of opiates as neuroimmunomodulatory agents and demonstrate that the effects of opiates on microglial cells may be influenced by the animal species from which the cells are derived.

Analgesics, Opioid↗
PubMed1997

Cloning and molecular characterization of CnTEF1 which encodes translation elongation factor 1alpha in Cryptococcus neoformans.

Degenerate PCR primers were synthesized based upon known translation factor 1alpha (TEF1) sequences. Touchdown PCR with these primers utilizing Cryptococcus neoformans strain M1-106 genomic DNA as template produced a DNA fragment containing a portion of CnTEF1. This DNA fragment was used as a hybridization probe to clone a cDNA version of CnTEF1 from C. neoformans strain B3501. Comparison of the genomic and cDNA nucleotide sequences revealed the presence of six introns in CnTEF1. The nucleotide sequence of CnTEF1 from these two strains of C. neoformans were 98% identical. Codon bias for most amino acids encoded by CnTEF1 was similar to that observed in Saccharomyces cerevisiae for highly expressed genes. This codon bias was also observed in the C. neoformans ACT gene. CnTEF1 encoded a protein (CnEF-1alpha) consisting of 459 amino acids with a calculated MW of 50.3 kDa from C. neoformans strain B3501. CnTEF1 from strain M1-106 encoded a protein with one additional aa. Both C. neoformans proteins possessed a high degree of identity throughout their length to fungal, human, and plant EF-1alpha proteins.

Amino Acid Sequence↗
PubMed1998

Signal transduction pathways regulating differentiation and pathogenicity of Cryptococcus neoformans.

The basidiomycetous yeast Cryptococcus neoformans is a human pathogen. Several phenotypes of this organism are defined as virulence traits including the polysaccharide capsule, melanin, and the ability to grow at 37 degreesC. The signaling pathways regulating the expression of these phenotypes and other important cellular processes are being defined on a molecular level. For example, the highly conserved signaling molecule calcineurin regulates high temperature growth in C. neoformans. A cryptococcal homolog of Saccharomyces cerevisiae STE12, the gene for a transcriptional regulator activated by the MAP kinase cascade, has also been identified. Additionally, the C. neoformans Galpha protein GPA1 and cAMP regulate mating, melanin production, encapsulation, and pathogenicity. This fungus is an excellent model to further dissect virulence-associated signaling pathways. The conserved role of Galpha proteins and cAMP-associated signaling pathways in fungal differentiation and pathogenicity is also reviewed.

Cryptococcosis↗
PubMed1999

Molecular analysis of the Cryptococcus neoformans ADE2 gene, a selectable marker for transformation and gene disruption.

Cryptococcus neoformans is an important fungal pathogen of man. The incidence of cryptococcal disease has increased dramatically in patients immunocompromised because of HIV infection, organ transplantation, or treatment with cytotoxic chemotherapy or corticosteroids. This organism is an excellent model for molecular dissection of fungal pathogenesis and virulence factors. Here we report the nucleotide sequence of the C. neoformans serotype D genomic ADE2 gene, which encodes a phosphoribosylaminoimidazole carboxylase required for purine biosynthesis. Importantly, this version of the ADE2 gene has been used as the selectable marker for virtually all gene disruptions by transformation and homologous recombination in C. neoformans. We compare the nucleotide and amino acid sequences of the ADE2 gene and product to other highly related adenine biosynthetic genes and enzymes from other yeasts and fungi. We also describe a series of convenient ADE2 cassettes for gene disruption construct preparation. Finally, we have identified the ade2 mutations in strains M001 and M049, adenine auxotrophic mutants derived from the serotype A strain H99. These mutant strains have served as recipients for targeted gene disruptions using the ADE2 gene. These studies should facilitate transformation and gene disruption approaches using the ADE2 selectable marker in this important human fungal pathogen.

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed2000

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.

Crosses, Genetic↗
PubMed2000

Development and characterization of a genetic linkage map of Cryptococcus neoformans var. neoformans using amplified fragment length polymorphisms and other markers.

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences. These markers were used to analyze 100 meiotic progeny. All markers were tested for distorted segregation with a goodness of fit test. Of the total of 181 markers, 148 showed balanced (1:1) segregation ratios. Segregation distortion was observed for 33 markers. Based on all the markers, a linkage map was generated that consists of 14 major linkage groups with 127 markers, several small linkage groups, and 2 linkage groups that consist only of highly skewed markers. The genetic distance of the linkage map is 1356.3 cM. The estimated total haploid genome size for C. neoformans var. neoformans was calculated using Hulberts method and yielded a map size of 1917 cM. The number of major linkage groups correlates well with the proposed number of 13 chromosomes for C. neoformans var. neoformans. Several genes, including CAP64, CnLAC, and the mating-type locus, were mapped, and their associations were consistent with published data. To date, 6 linkage groups have been assigned to their corresponding chromosomes. This linkage map should provide a framework for the ongoing genome sequencing project and will be a useful tool for studying the genetics and pathogenicity of this important medical yeast.

Chromosome Mapping↗
PubMed1996

Peptide libraries define the fine specificity of anti-polysaccharide antibodies to Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated fungus that causes a life-threatening meningoencephalitis in patients with AIDS. Monoclonal antibodies to the capsular glucuronoxylomannan can modulate the infection in mice, but the epitopes on this complex polysaccharide recognized by protective and non-protective antibodies have not been defined. We have used 2H1, one of our most protective antibodies, to screen phage display peptide libraries for peptide mimotopes that would allow us to explore the fine specificity of anti-cryptococcal polysaccharide antibodies. Hexa- and decapeptides have been identified with sequence homologies that define four motifs: 1, (E)TPXWM/LM/L; 2, W/YXWM/ LYE; 3, DWXDW; and 4, (Ar)WDGQ(Ar). Peptides representing these motifs compete with each other for a shared binding site that overlaps the polysaccharide binding site. Motifs 1 and 2 confer high affinity binding, and PA1, which displays a motif 1 peptide with the sequence LQYTPSWMLV, binds to 2H1 with a Kd of 295 nM. Analysis of the interaction between the 2H1 binding peptides and 24 structurally related anti-polysaccharide antibodies reveals a complex pattern of reactivity that strongly suggests binding to or close to the complementary determining regions. Furthermore, those antibodies that have been shown to have different specificity, and in some cases different protective potential, do not bind any of the peptides selected by the protective 2H1 antibody. This study shows that peptide mimotopes for a complex microbial polysaccharide can be identified by screening phage peptide libraries and demonstrates the usefulness of such peptides in analyzing closely related interactive sites of proteins in general and of antibodies in particular.

Amino Acid Sequence↗
PubMed1997

The three-dimensional structures of a polysaccharide binding antibody to Cryptococcus neoformans and its complex with a peptide from a phage display library: implications for the identification of peptide mimotopes.

The three-dimensional structure of 2H1, a protective monoclonal antibody to Cryptococcus neoformans, has been solved at 2.4 A resolution, in both its unbound form and in complex with the 12 amino acid residue peptide PA1 (GLQYTPSWMLVG). PA1 was previously identified as a potential mimotope of the cryptococcal capsular polysaccharide by screening of a phage display peptide library. Peptide binding is associated with only minor rearrangements of some side-chains and a small shift in the H2 loop of the antibody. The peptide assumes a tightly coiled conformation consisting of one inverse gamma-turn and one type II beta-turn that serves to place the entire peptide motif, consisting of ThrP5, ProP6, TrpP8, MetP9 and LeuP10, into a depression in the antibody combining site. A small number of H-bonds between peptide and antibody contribute to the affinity and specificity. Poor steric complementarity between PA1 and the antibody heavy chain along with the fact that the majority of the interactions between 2H1 and PA1 involve van der Waals interactions with the light chain may explain why this peptide acts as only a partial mimotope of the capsular polysaccharide epitope.

Amino Acid Sequence↗
PubMed2000

A pre-existing infection by Mycobacterium avium subsp. avium modulates anti-Cryptococcus neoformans and anti-Candida albicans activities in human macrophages.

Mycobacterium avium is a facultative intracellular microorganism, able to survive and multiply within mammalian macrophages by circumventing antimicrobial mechanisms. In this study we hypothesize that pre-existing M. avium infection could result in macrophage superinfections by other microorganisms. We found that 24 h after ingestion of M. avium at a low multiplicity of infection, macrophages are unable to efficiently produce superoxide anions when over-stimulated with phorbol esters, and that the generation of oxidative burst is only partially restored 72 h after bacteria ingestion. We also demonstrate that intracellular killing of Cryptococcus neoformans is markedly impaired in human macrophages that have previously ingested M. avium (but not other bacteria such as Escherichia coli). This inhibitory effect is observed with live mycobacteria, but not when heat-inactivated bacteria are ingested. In contrast, when Candida albicans is given to macrophages instead of C. neoformans, an enhancement of intracellular killing is observed, suggesting that cytocidal mechanisms other than respiratory burst are involved in the anti- Candidacidal activity of macrophages.

Candida albicans↗
PubMed1992

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants.

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.

Base Sequence↗