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PubMed1996

Structure of the O-deacetylated glucuronoxylomannan from Cryptococcus neoformans Cap70 as determined by 2D NMR spectroscopy.

Cryptococcus neoformans, an opportunistic pathogen, is the fourth leading cause of death among AIDS patients. The yeast's capsule is a major virulence factor, and serotype is related to the chemical structure of glucuronoxylomannan (GXM), its capsular polysaccharide. The GXM from Cap70, a hypocapsular mutant of serotype D isolate B-3501, was investigated by chemical analysis and 2D NMR spectroscopy. The assignment of 1H and 13C chemical shifts for the O-deacetylated polysaccharide was accomplished from the analysis of DQF-COSY, TOCSY, and gradient-enhanced HSQC spectra. The sequence and linkage positions of glycosyl residues were determined by NOESY and ROESY spectra. Two repeating polysaccharide components were identified as having the following structures in approximately equal proportions: [formula: see text] It is not known if these repeating units comprise a single or two separate polymer chains. Pentasaccharide 2 has been known to be the major GXM polymer of B-3501 and other serotype D isolates. Hexasaccharide 1 is identified for the first time although it has subsequently been identified in other C. neoformans isolates. The presence of 1 in the GXM of Cap70 is consistent with the extra xylose found relative to that in isolate B-3501. The mannose:xylose:glucuronic acid:O-acetyl molar ratio of Cap70 GXM is 3.00:1.73:0.78:1.75, while the same ratio for B-3501 and other serotype D isolates is approximately 3.00:1.00:0.80:1.75. Methylation analysis confirmed that the GXM of Cap70 contains unsubstituted, monosubstituted (2-linked), and disubstituted (2- and 4-linked) mannose in a ratio of 0.87:1.75:0.38. Dot blot immunoassay indicates that Cap70 is a serotype D isolate like its parent strain.

Acetylation↗
PubMed1995

Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients.

Azole antifungal compounds are important in the treatment of Cryptococcosis, a major cause of mortality in AIDS patients. The target of the azole drugs is P450 mediated sterol 14 alpha-demethylase. We have investigated the P450 system of Cryptococcus neoformans with respect to azole tolerance observed in clinical isolates which were obtained following the failure of fluconazole therapy. The clinical failure was correlated with in vitro tolerance of azole antifungal when compared to wild-type strains. The microsomal P450 system was typical of yeast and fungi and fluconazole tolerance was not associated with defective sterol biosynthesis. The strains had slightly elevated P450 content and slightly reduced azole levels in the cells, but a clear cause for resistance was the increased level of drug needed to inhibit the sterol 14 alpha-demethylase in vitro.

AIDS-Related Opportunistic Infections↗
PubMed1995

Monoclonal antibody mediated capsular reactions (Quellung) in Cryptococcus neoformans.

Capsular reactions ('Quellung') visible by light microscopy were observed when capsule-binding monoclonal antibody (mAb) was added to Cryptococcus neoformans yeast cells. Capsular reactions were observed with capsule-binding IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA mAbs. Scanning electron microscopy revealed that mAb binding produced a structural change in the fibrillar network of the capsule. The occurrence of capsular reactions with mAbs indicate that this phenomenon can be produced by the binding of antibody to a single epitope.

Animals↗
PubMedDate not supplied

Murine monoclonal antibodies recognizing a non-capsular antigen that distinguishes between Cryptococcus neoformans var. neoformans and C. neoformans var. gattii.

A panel of 4 monoclonal antibodies (mabs) of the IgG1 subclass have been made against a cytoplasmic antigen of Cryptococcus neoformans. Mab 4E2 recognized isolates of C. neoformans var. gatti by enzyme-linked immunosorbent assay (ELISA), whilst the other antibodies did not recognize these antigens. By Western blot 4E2 recognized determinants at 110-125, 65-70, 45-50 and 36-38 kDa. Mabs 9E6, 7C7 and 5D9 recognized bands at 36-38 and approximately 30 kDa. All 4 mabs (4E2, 9E6, 7C7 and 5D9) recognized both non-encapsulated and encapsulated isolates of C. neoformans var. neoformans by ELISA, and in addition showed reactivity to only the cytoplasm and cell membrane of yeasts by immunofluorescence. Mab 7C7 recognized antigens of the closely related fungus Trichosporon beigelii by ELISA but did not recognize any other fungal antigens. The other 3 mabs showed no recognition of T. beigelii or any other fungal pathogens tested.

Animals↗
PubMed1990

Ecology, life cycle, and infectious propagule of Cryptococcus neoformans.

Cryptococcus neoformans is a biotrophic smut-like fungus, and the epidemiology of cryptococcosis can mainly be explained by exposure to an infective aerosolised inoculum. For C neoformans var gattii it is postulated that the principal infectious propagule is the basidiospore and that exposure to Eucalyptus camaldulensis, the host tree, is required to initiate infection in man and animals. C neoformans var gattii may have been exported from Australia by infected seeds of E camaldulensis containing dormant dikaryotic mycelium of the fungus. For C neoformans var neoformans both the basidiospore and desiccated encapsulated yeast cells are postulated to act as infectious propagules, the basidiospores showing a seasonal distribution in association with an as yet unidentified host plant, and the encapsulated yeast cells dispersed from accumulations of dried bird (mainly pigeon) droppings which act as a year-round vector.

Algorithms↗
PubMedDate not supplied

Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms.

Acetylation↗
PubMed1995

Human microglia mediate anti-Cryptococcus neoformans activity in the presence of specific antibody.

The interaction of the opportunistic fungus Cryptococcus neoformans with human microglia was studied in vitro in the presence and absence of capsule binding antibody. In the absence of capsule binding antibody there was little or no phagocytosis. Addition of the murine monoclonal antibody (mAb) 2H1 (IgG1, kappa) to the capsular glucuronoxylomannan (GXM) produced a dose-dependent enhancement of C. neoformans phagocytosis by microglia. Phagocytosis resulted in marked inhibition of fungal proliferation. Microglial antifungal activity was studied by colony forming unit assay, L-[3H]leucine incorporation assay, and phase contrast microscopy. At microglia: C. neoformans ratios of 10:1 to 80:1 fungal growth was reduced by 61-95%. Inhibitors of nitric oxide synthase and reactive oxygen intermediates did not prevent antifungal activity mediated by human microglia. Transmission electron microscopic studies revealed that although some internalized yeast cells were killed, the majority were intact consistent with fungistasis. Human microglia cells are potent effector cells against C. neoformans in vitro in the presence of specific antibody. Enhancement of microglial activity in vivo by opsonins may be a useful therapeutic strategy.

Antibodies, Fungal↗
PubMed1985

Molecular expression of xylanase gene in Cryptococcus albidus.

In the yeast Cryptococcus albidus, the utilization of xylan as compared to xylose requires at least an inducible endoxylanase enzyme, secreted in the culture medium. The endoxylanase induction was monitored by immunoprecipitation of in vivo and in vitro synthesized products. The mature endoxylanase is a highly glycosylated enzyme with an apparent molecular weight of 48 000. Upon chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight was reduced to 40 000. Addition of tunicamycin to the culture medium resulted in the synthesis of a modified polypeptide having a molecular weight of 40 000. Poly(A)-containing RNA isolated from the yeast was translated in the rabbit reticulocyte protein-synthesizing system. The appearance of a translatable xylanase mRNA was observed in xylan-grown cells but not in xylose-grown cells. The polypeptide identified as xylanase had a molecular weight of 44 000. This suggests that the xylanase is synthesized as a precursor, containing a peptide signal sequence of 35 residues.

Animals↗
PubMed1996

Purification and properties of D-aspartate oxidase from Cryptococcus humicolus UJ1.

D-Aspartate oxidase (EC 1.4.3.1), which is highly specific to D-aspartate, was inducibly produced by a yeast strain which was isolated from soil and identified as Cryptococcus humicolus UJ1. The enzyme was purified to homogeneity as indicated on SDS-polyacrylamide gel electrophoresis. The molecular mass of the monomer subunit was determined to be 40 kDa. The native enzyme was suggested to be a homotetramer by its behavior on gel filtration. The enzyme was shown to be a flavoprotein by its absorption spectral properties, and the flavin was found to be tightly, but not covalently, bound FAD. The purified preparation had a specific activity of 76.1 mumol/min per mg protein with D-aspartate as substrate. Optimum pH was 7.5 and optimum temperature was around 35 degrees C. D-Glutamate was a very poor substrate for the enzyme. N-Methyl-D-aspartate was better than D-glutamate as substrate but markedly poorer than D-aspartate. Malonate was the most effective competitive inhibitor of the compounds tested. The N-terminal amino-acid sequence of the enzyme showed a significant homology with those of D-aspartate oxidases from beef kidney and Octopus vulgaris and those of D-amino-acid oxidases from various sources.

Amino Acid Oxidoreductases↗
PubMed1996

Preclinical efficacy of a glucuronoxylomannan-tetanus toxoid conjugate vaccine of Cryptococcus neoformans in a murine model.

The encapsulated yeast, Cryptococcus neoformans, causes life-threatening meningoencephalitis in immunocompromised humans, especially in AIDS patients. Fatality and relapse rates remain quite high despite aggressive therapy. A conjugate vaccine composed of the cryptococcal capsular glucuronoxylomannan covalently coupled to tetanus toxoid (GXM-TT) was constructed and evaluated. The vaccine elicited high levels of capsular antibodies in mice by active and passive immunizations and conferred 70-80% protection against a moderate challenge with 10(3) C, neoformans. Monitoring of serum GXM and anti-GXM antibody levels and of incidence of cryptococcal isolation from various organs of mice suggested that presence of vaccine-induced antibodies during the first 4-6 weeks of infection is critical for clearance of cryptococci from various organs, for limiting serum GXM titers from reaching immunosuppressive levels and ultimately for survival. GXM-TT is the first defined fungal vaccine to confer antibody-mediated protection against a systemic mycosis in an animal model. GXM-TT is being evaluated for safety and immunogenicity in healthy and HIV-infected human volunteers at the National Institutes of Health.

Animals↗
PubMed1989

Stimulation of xylanase synthesis in Cryptococcus albidus by cyclic AMP.

Cryptococcus albidus secretes a xylanase when induced by xylan or beta-methylxyloside, a non-metabolizable inducer, and production of the enzyme is repressed by xylose. The effect of exogenous cAMP on xylanase production was tested under different growth conditions. The cAMP elicited a 1.5 to 2 fold increase in xylanase production during the induction by xylan and B-methylxyloside but did not relieve the repression observed during growth on xylose. Cyclic AMP also affected the growth rate of the cells and did not modulate the activity of pure xylanase in vitro. A 15-nucleotide sequence located upstream from the xylanase gene could be part of a cAMP regulatory sequence.

Base Sequence↗
PubMed1992

Cloning the Cryptococcus neoformans TRP1 gene by complementation in Saccharomyces cerevisiae.

We have cloned the phosphoribosyl anthranilate isomerase (PRAI)-encoding gene (TRP1) of Cryptococcus neoformans by genetic complementation in Saccharomyces cerevisiae. Sequence analysis of this gene revealed it to be 939 bp in length, and without known promoter or termination sequences. Unlike some of the filamentous fungi, where PRAI enzymatic activity is controlled by a trifunctional gene product, the C. neoformans PRAI appears to be unifunctional. PRAI of C. neoformans exhibits 39% amino acid (aa) sequence identity compared to the S. cerevisiae counterpart. The TRP1 gene of C. neoformans maps to different size chromosomes in strains with different serotypes. The cloning of this gene for vector constructions, and the demonstration that S. cerevisiae can be used as a surrogate for C. neoformans gene expression, should help with the molecular studies of this significant fungal pathogen in our increasing immunocompromised population.

Aldose-Ketose Isomerases↗
PubMed1992

Cloning and expression in Escherichia coli of a xylanase-encoding gene from the yeast Cryptococcus albidus.

In the yeast, Cryptococcus albidus, a comparison between the sequence of the xylanase (XLN)-encoding chromosomal gene (XLN) and the cDNA sequence reveals the presence of seven introns, ranging in length from 51 to 69 bp. One of their 5' splice site sequences is similar to the consensus sequence for yeast, while the other six resemble the consensus sequence for higher eukaryotes. Their 3' end splice site sequences are representative of the conserved sequence found in eukaryotes. Their putative branching point sequences are different from the well-known conserved sequence, 5'-TACTAAC, observed in yeast, but again resemble the mammalian one. The cDNA encoding XLN is expressed by Escherichia coli, under the control of the lacZ promoter. The gene product remains inside the cell and has a molecular size of 40 kDa, which matches the size of the nonglycosylated protein. When compared to the glycosylated enzyme, the nonglycosylated XLN from E. coli shows twofold less affinity for substrate and its Vmax is 100-fold lower. Moreover, the nonglycosylated XLN only acts on large xylan polymers and very slightly on xylohexaose.

Base Sequence↗
PubMed1992

Secretion of a Cryptococcus albidus xylanase in Saccharomyces cerevisiae.

The xylanase(XLN)-encoding gene(XLN) of Cryptococcus albidus and its cDNA were each inserted into the vector, pVT100, for expression in Saccharomyces cerevisiae. Expression was under the control of either their own promoter or the gene encoding alcohol dehydrogenase (ADH1) promoter. Yeast transformed with plasmids containing the cDNA of the structural XLN gene and the XLN promoter produced active extracellular XLN when grown with galactose as carbon source. However, with glucose as carbon source, XLN was repressed. Using the ADH1 promoter, which is stimulated by glucose, XLN was secreted into the culture medium. In both cases, the secreted 48-kDa enzyme corresponded to the native XLN produced by C. albidus. With the plasmid bearing the genomic XLN gene, there was transcription, but the seven introns interrupting XLN were not spliced out by S. cerevisiae and no enzyme was produced.

Alcohol Dehydrogenase↗
PubMed1994

Characterization of the CNRE-1 family of repetitive DNA elements in Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans contains 10-20 dispersed repetitive elements that hybridize to clone CNRE-1.0. Screening of a genomic library with probes derived from CNRE-1.0 identified five phages with restriction maps that overlapped CNRE-1.0 and three additional phages that belonged to two distinct groups. Sequencing of internal 3.5-kb SstI fragments from two CNRE-1-like elements revealed 95% homology, as well as a conserved open reading frame. A PCR-RFLP assay was developed that can distinguish different subfamilies of CNRE-1-like elements.

Base Sequence↗
PubMed1994

Cloning, sequence analysis and expression of the gene encoding imidazole glycerol phosphate dehydratase in Cryptococcus neoformans.

A cDNA from Cryptococcus neoformans, encoding imidazole glycerol phosphate dehydratase (IGPD), was isolated by complementation of a his3 mutant strain of Saccharomyces cerevisiae. The C. neoformans HIS3 cDNA encodes an approx. 22-kDa protein with a high degree of amino-acid sequence similarity to IGPDs from ten other microorganisms, as well as Arabidopsis thaliana. Most striking are two conserved HHXXE regions and several conserved His, Asp and Glu residues. The cDNA was engineered for expression in Escherichia coli and an approx. 26-kDa protein was identified by SDS-PAGE. DNA and N-terminal sequence analyses confirmed that this protein was C. neoformans IGPD. Furthermore, IGPD assays of crude extracts from IGPD-producing E. coli cells demonstrated that the C. neoformans protein was catalytically active.

Amino Acid Sequence↗
PubMed1994

Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans.

The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans (Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35,844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene. The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the Km values for dUMP and CH2H4.folate were 2.7 +/- 0.5 microM and 38.2 +/- 2.5 microM, respectively, and the kcat was 5.1 s-1. The enzyme was stable upon storage at -80 degrees C in Tris.HCl pH 7.4 and thiol.

Amino Acid Sequence↗
PubMed1995

Structure of the ubiquitin-encoding genes of Cryptococcus neoformans.

Cryptococcus neoformans (Cn) contains two ubiquitin (UBI)-encoding genes located on separate chromosomes. The UBI1 gene consists of UBI fused to a 53-amino-acid (aa) tail and is 95% identical to the Saccharomyces cerevisiae (Sc) UBI1 which codes for an UBI-CEP52 ribosomal protein fusion. UBI4 is a polyubiquitin gene that contains five UBI repeats. The UBI4 aa sequences differ from Sc UBI by a single aa. UBI1 contains two introns in the UBI-encoding portion and two introns in the tail. Single introns are present in three of the repeats in UB14 and are located at the same positions as those in UBII. There was also an average of 15% nt differences among UBI repeats. The results provide evidence of extensive recombination and/or conversion events between repeated genes in Cn.

Amino Acid Sequence↗