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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Development of a transformation system for the fungal human pathogen Cryptococcus neoformans is an important prerequisite for the identification of genes involved in virulence. It has previously been reported that low-efficiency transformation can be achieved by using the cloned C. neoformans URA5 gene and ura5 mutants. The introduction of linearized URA5 vectors into C. neoformans resulted in unstable transformants which apparently harbored linear extrachromosomal DNA molecules. In this paper, the nature of these molecules is confirmed to be linear by exonuclease digestion. Recovery of the extrachromosomal DNA in Escherichia coli and sequence analysis demonstrates that repeats characteristic of telomeric DNA have been added to the ends of the introduced DNA. The recovered plasmids are capable of transforming at much higher efficiencies either in the supercoiled state (up to 200 transformants per microgram) or the linear state (up to 90,000 transformants per microgram).
Mol Cell Biol 1992 Jun
PMID:Isolation of telomerelike sequences from Cryptococcus neoformans and their use in high-efficiency transformation. 158 69

Cryptococcus neoformans is a pathogenic yeast causing disease predominantly in immunosuppressed patients. C. neoformans is acquired by the pulmonary route, where the alveolar macrophage (AM) is a resident mechanism of host defense. The ability of rat AM to be activated by products of the immune response for enhanced anticryptococcal effect has not previously been demonstrated. Rat AM could be activated in vitro for anticryptococcal activity by medium conditioned by concanavalin A-stimulated splenic lymphocytes. A monoclonal antibody that neutralizes interferon-gamma (IFN-gamma) inhibited the macrophage-activating activity of lymphokine-containing medium (LCM). Further, recombinant IFN-gamma activated AM for anticryptococcal activity. The concentration of IFN-gamma in LCM, determined by enzyme-linked immunosorbent assay, was equivalent to the range of concentrations of recombinant IFN-gamma which activated AM. Thus, IFN-gamma was necessary and sufficient for optimal macrophage activation by medium conditioned by proliferating lymphocytes. Lipopolysaccharide could not enhance the anticryptococcal activity produced by optimal concentrations of LCM or IFN-gamma but did augment the effects of submaximal stimulation. Both LCM and recombinant IFN-gamma increased the percentage of macrophages with cell-associated cryptococcus, suggesting that activation of AM enhanced the adhesion or uptake of cryptococcus. We speculate that inadequate availability of lymphokines such as IFN-gamma may result in the immunodeficient state in hosts unable to generate an appropriate response to cryptococcal antigens. Administration of lymphokines such as IFN-gamma to immunosuppressed hosts might circumvent the defect in cell-mediated immunity.
Am J Respir Cell Mol Biol 1991 Jul
PMID:Interferon-gamma activates rat alveolar macrophages for anticryptococcal activity. 190 86

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.
Mol Immunol
PMID:Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces. 206 24

A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.
Mol Cell Biol 1990 Sep
PMID:Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation. 220 94

Three monoclonal antibodies (MAbs) (BA4, BD1, CD6) reacted with Cryptococcus neoformans capsular glucuronoxylomannan (GXM) polysaccharide showing distinctive patterns against four serotypes as revealed by enzyme immunoassay (EIA), dot EIA, and immunofluorescence. Immunoelectron microscopy (IEM) was used to characterize binding sites for the MAbs on the C. neoformans capsule. All three MAbs bound to the capsule of serotype A strains 9104 and 9759. Differences in the intensity of binding to the two serotype A strains could not be explained by capsule diameter. The MAb BA-4 IgM bound well to 9759 (large capsule) and poorly to 9104 (small capsule), whereas MAb BD-1 (IgG-1) bound well to strain 9104 and poorly to strain 9759. Spurr's embedment inactivated the BA-4-binding epitopes in the C. neoformans 9759 capsule, but did not inactivate the ones that bound to BD-1. The epitopes recognized by BA-4 were different than the BD-1-binding determinants. The MAb CD-6 bound to a cytoplasmic precursor of capsular GXM. CD-6 (IgG) stained the capsule, cell wall, and cytoplasm of both C. neoformans tester strains. Competitive binding experiments were conducted. Single immunogold labelling showed that BD-1 inhibited the binding of BA-4, but not vice versa. The interaction between CD-6 and BA-4 resulted in a reciprocal inhibition. Double-labelling experiments showed reciprocal inhibition between BA-4 and each of the IgG MAbs. These MAbs are directed against capsular polysaccharide or its intracellular precursor. None of the MAbs stained C. neoformans cap 67, an acapsular mutant that does not contain GXM.
Mol Cell Probes 1989 Dec
PMID:Immunoelectronmicroscopic characterization of monoclonal antibodies (MAbs) against Cryptococcus neoformans. 248 37

Antibodies to Cryptococcus neoformans Type D have been raised in rabbits. The partially purified antibody pool was fractionated by affinity-chromatography, and it was shown that antibodies to the capsular polysaccharide of Type D were present in the pool. Neither methyl alpha-D-mannopyranoside, nor D-mannose were inhibitors of the binding of Type D polysaccharide to the antibody-pool. Thus it appears that the serum contains antibody populations which are specific for terminal side-chain carbohydrate moieties of the immunizing antigen.
Mol Immunol 1983 Apr
PMID:Anticryptococcal type D antibodies raised in rabbits. 619 Dec 5

A murine pulmonary infection model utilizing intratracheal inoculation of Cryptococcus neoformans was used to analyze cytokines produced in response to opportunistic pathogens acquired via the respiratory tract. The specific question asked was whether early cytokine secretion in lung-associated lymph nodes (LALN) would predict whether this organism would be cleared from the lung. Lung colony-forming units (CFU) were analyzed in two strains of mice over 12 wk, and lung clearance was found to be strain dependent. C.B-17 mice reduced their lung CFU burden between day 7 and day 14 of infection, had significantly higher in lung CFU than C.B-17 mice. The capacity of cells from lungs and LALN to secrete cytokines was significantly different between the strains when assessed at day 7 and day 14 after inoculation. When compared with sensitive C57BL/6 mice 7 days after infection, resistant C.B-17 mice demonstrated (1) increased interferon-gamma secretion by LALN cells in vitro in response to media alone, heat-killed cryptococci, and the T cell mitogen concanavalin A and (2) increased interleukin (IL)-2 secretion by both LALN and lung cells in response to concanavalin A. IL-4 and IL-10 were comparable or undetectable in both mouse strains, whereas IL-5 was significantly higher in all lung cell cultures of C57BL/6 mice. Thus, an early regional Th1 immune response in C.B-17 mice correlated with resistance to the organism, whereas the absence of this response in C57BL/6 mice correlated with susceptibility.
Am J Respir Cell Mol Biol 1995 Oct
PMID:Early cytokine production in pulmonary Cryptococcus neoformans infections distinguishes susceptible and resistant mice. 754 79

Increasing evidence indicates that T cell-dependent, interferon gamma (IFN gamma)-induced activation of murine macrophages and nitric oxide (NO) production plays an important role in host defenses against many microorganisms. A role for this mechanism in pulmonary defenses against infectious agents has not been examined. Previous studies demonstrated that both CD4 and CD8 T cells were required for lung clearance of encapsulated Cryptococcus neoformans (Cne). The current studies investigated whether IFN gamma-induced NO production was involved in the protective T cell-mediated immune response against Cne. Intratracheal inoculation of a low-virulence strain of Cne into mice resulted in an infection that was progressively cleared in immunocompetent C.B-17, but not severe combined immunodeficient (SCID) mice. The onset of Cne lung clearance in immunocompetent mice coincided with a marked increase in inflammatory cells in the lung, local expression of IFN gamma-inducible nitric oxide synthase (iNOS) messenger RNA (mRNA), and an increase in systemic NO production as measured by urinary nitrate excretion. None of these changes were observed in infected SCID mice. Inflammatory lung cells isolated from Cne-infected C.B-17 mice inhibited the growth of endogenous Cne in vitro by a NO-dependent mechanism. Moreover, lung clearance of Cne in immunocompetent mice was blocked by treatment with (1) antibody to IFN gamma, which blocked iNOS gene expression and NO production, or (2) the arginine analogue, NGmonomethyl-L-arginine (MMA), which only blocked NO production. However, neither anti-IFN gamma nor MMA treatment decreased the numbers or types of recruited inflammatory cells. Thus, these studies demonstrated that, although recruitment of effector cells was required, it was not sufficient to initiate clearance of Cne from the lung. Rather, an IFN gamma-induced effector mechanism, i.e., NO production, was also required.
Am J Respir Cell Mol Biol 1995 Jul
PMID:A role for gamma interferon-induced nitric oxide in pulmonary clearance of Cryptococcus neoformans. 759 35

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.
Mol Cell Biol 1994 Jul
PMID:Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. 800 87

The contribution of human alveolar macrophages (AM) from normal subjects in Cryptococcus neoformans infection was investigated. AM were able to efficiently phagocytize the fungus after opsonization, but killing activity did not occur at an effector-to-target ratio of 10:1 in a 6-h incubation since there was an inhibition of phagosome-lysosome fusion. Moreover, the role of AM as antigen-presenting cells was investigated. Cryptococcus-laden AM were co-cultured with autologous T lymphocytes and lymphoproliferation was determined; a massive blastogenic response of alpha/beta TCR-bearing T lymphocytes was observed. The response started after 1 day of co-culture and was triggered and regulated by IL-1 produced by AM in response to C. neoformans. Finally, the antigen-presentation process was associated with HLA class II DR molecules. This finding suggests that AM play a key role in the lung as antigen-presenting cells and, through the secretion of IL-1, regulate proliferation and activation of T lymphocytes, which are important in mediating pulmonary clearance. We speculate that in immunodepressive conditions, the impairment of AM functions could contribute to the spread of C. neoformans infection from the lung.
Am J Respir Cell Mol Biol 1994 Aug
PMID:Role of human alveolar macrophages as antigen-presenting cells in Cryptococcus neoformans infection. 804 74


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