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PubMed1985

Ultrastructure of Cryptococcus neoformans in the cerebrospinal fluid of a patient with cryptococcal meningitis.

The ultrastructure of Cryptococcus neoformans in the cerebrospinal fluid, which was obtained from a patient suffering from systemic lupus erythematosus and cryptococcal meningitis, before treatment and on the 10th and 20th day after the start of treatment was studied. Numerous cryptococci were detected in their cerebrospinal fluid before treatment. However, in most of them their biological activity was low and their organelles were not clear. A few yeasts preserved well their organelles. Such cells showed a tendency to develop vesicular membrane structures (lomasomes) touching the internal part of the cell wall. In the cerebrospinal fluid on the 20th day all of the yeasts were dead, even though numerous yeasts were observed by an India-ink method. At this time no colonies were recovered from the fluid.

Adult↗
PubMed1979

Chemotaxigenesis and activation of the alternative complement pathway by encapsulated and non-encapsulated Cryptococcus neoformans.

In the presence of serum, whole cells of encapsulated and non-encapsulated Cryptococcus neoformans generated a chemotactic response by neutrophils. Heat inactivation of serum ablated all chemotactic activity. Cryptococcal polysaccharide was not chemotaxigenic. Assays for alternative complement pathway activation such as depletion of alternative complement pathway factor B or electrophoretic conversion of factor B closely paralleled chemotaxis assays. Cells of encapsulated and non-encapsulated C. neoformans activated the alternative complement pathway, whereas cryptococcal polysaccharide was inactive. Failure of the capsular material to activate the alternative pathway was not due to serotype specificity because polysaccharide of several serotypes failed to achieve activation. The results suggest that chemotaxigenesis and alternative complement pathway activation are functions of the yeast cell wall. The results support our proposal that the cryptococcal capsul does not prevent potential opsonins from reaching binding and activation sites at the yeast cell wall or the release of biologically active soluble cleavage products into the surrounding medium; however, cell wall-bound cleavage products remain bound to the cell wall beneath the capsule. Therefore, they are unable to participate as opsonins in phagocytosis.

Animals↗
PubMed2005

A new hexose transporter from Cryptococcus neoformans: molecular cloning and structural and functional characterization.

We carried out a screen for Cryptococcus neoformans genes involved in resistance to copper ion toxicity and identified a new hexose transporter (Hxt) gene, HXT1. Hxt1 consists of 520 amino acids and functions to transport hexoses such as glucose. Although Hxt1 conferred copper resistance to Saccharomyces cerevisiae, disruption of the HXT1 gene showed that Hxt1 is not necessary for copper resistance. In virulence tests, an hxt1 mutant strain showed 12% less phenoloxidase activity than the wild-type strain, and no difference in the ability to form melanin was identified. In addition, the hxt1 mutant strain showed virulence similar to that of the wild-type strain in experiments with Caenorhabditis elegans. However, the hxt1 mutant strain generated larger capsules than were generated by the wild-type strain. Thus, Hxt1 appears to be involved in capsule formation.

Animals↗
PubMed1998

Effect of antibody to capsular polysaccharide on eosinophilic pneumonia in murine infection with Cryptococcus neoformans.

The effect of the murine IgG1 monoclonal antibody (MAb) 2H1, which binds to Cryptococcus neoformans glucuronoxylomannan (GXM), on pulmonary infection in immunocompetent C57Bl/6 mice was examined. C57Bl/6 mice develop eosinophilic pneumonia in response to pulmonary cryptococcal infection. Survival, organ fungus burden, serum anticapsular antibody levels, and histopathology by light and electron microscopy were studied. MAb administration prior to infection prolonged survival without reducing the number of yeast in the lung or extrapulmonary sites. Compared with uninfected mice, occasional control and MAb-treated mice produced more IgM antibody to GXM or low levels of GXM-binding IgG1, IgG2b, or IgG3 antibodies. MAb-treated mice had fewer granules per eosinophil, indicating alteration in eosinophil physiology or degranulation (or both). Our results provide additional evidence that antibody administration can produce quantitative and qualitative changes in the inflammatory response to a pathogen.

Animals↗
PubMed1982

Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans.

Stable mutants with reduced capacity to produce capsules were isolated from suspensions of Cryptococcus neoformans after treatment of the wild type with a mutagen. The mutants could be assigned one of two phenotypes, hypocapsular or acapsular. Hypocapsular mutants were immunochemically and physicochemically indistinguishable from the wild type, whereas acapsular mutants lacked a major capsular antigen and a negatively charged exterior. In genetic analysis, the mutant trait segregated as a Mendelian gene (1:1) when random basidiospores from an outcross were studied, and analysis of products of single meiotic events from outcrossed mutants was likewise consistent with meiotic segregation. Two-factor crosses yielded the expected four classes of progeny, with recombinants equal to parentals. We concluded that chromosomal genes are responsible for synthesis of the cryptococcal capsule and that random basidiospore analysis represents a useful technique for genetic analysis in this species.

Antigens, Fungal↗
PubMed1996

A vitamin-free minimal synthetic medium for Cryptococcus neoformans.

The use of a simple synthetic medium is essential for study on the growth and physiology of Cryptococcus neoformans. In the present study, a minimal synthetic liquid medium (MSM) was tested for the growth of 23 C. neoformans strains. This medium contained a low concentration of glucose, ammonium sulphate and inorganic salts with a pH value of 4.5, but no amino acids or vitamins. The strains were starved for 4 days to eliminate nutrients which might have been carried over from their pre-culture medium. Then, they were inoculated in the MSM as an initial OD of 0.020 at 550 nm and incubated at 37 degrees C for 20 days. Cell growth was generally monitored daily by measuring the absorbance at 550 nm. The medium supported the growth of the strains tested and gave an average final OD of 0.500. The results obtained indicate that C. neoformans may be autotrophic with respect to vitamins and in particular to thiamine. The MSM medium is easy to prepare and store. It is highly reproducible and useful for studies on the growth and physiology of C. neoformans.

Cryptococcus neoformans↗
PubMed2003

Molecular analysis of Cryptococcus neoformans mitochondrial cytochrome b gene sequences.

Mitochondrial cytochrome b genes (cyt b) of 40 strains of Cryptococcus neoformans were partially sequenced to determine the genetic relations. With the exception of the type strain of C. neoformans var. neoformans, all strains contained introns in their sequences. Analysis of 386 bp of coding sequence from each strain under investigation revealed a total of 27 (6.99%) variable nucleotide sites and categorized isolates of C. neoformans into nine cyt b types. C. neoformans var. gattii included cyt b types I to V, and C. neoformans var. neoformans comprised types VI to IX. cyt b types were correlated with serotypes. All strains with cyt b types I, IV, and V were serotype B. All other strains except IFM 5878 (serotype B) with cyt b types II and III were serotype C. Serotype D strains had cyt b types VI and IX, and serotype A strains were cyt b type VIII. Of four serotype AD strains, one was cyt b type VII and the remaining three were type VIII. The phylogenetic tree based on deduced amino acid sequences divided the strains only into C. neoformans var. neoformans and C. neoformans var. gattii. These results indicate that cyt b sequences are effective for DNA typing as well as phylogenetic analysis of C. neoformans.

Base Sequence↗
PubMedDate not supplied

[Demonstration of Cryptococcus neoformans in a fatal meningeal disease in the Congo].

Cryptococcus neoformans, mainly widespread in Occidental Europe, America and South East of Asia, was rarely described in African pathology. In Central Africa, it was isolated several times in Zaïre. All reported cases concern young people and present no associated pathology. A first fatal case, looking like an acute meningitis, was described in 1959, in a 18 years old Congolese girl. This second case discovered in Brazzaville in a 37 years old young man was a fatal encephalitis after a primary pneumopathy.

Adult↗
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↗
PubMedDate not supplied

Effects of three azole derivatives on the lipids of different strains of Cryptococcus neoformans.

The comparative effects of ketoconazole, itraconazole and fluconazole on the lipids of four Cryptococcus neoformans strains were investigated. Quantitative analysis of lipids and sterols was completed, as well as qualitative analysis of sterols by thin-layer chromatography and by the ultraviolet spectrum. Growth of the cryptococcal isolates in the presence of the azoles derivatives concentrations below the minimum inhibitory concentration resulted in significant alterations in the lipid and sterol contents as compared with the control values. Furthermore, lanosterol was detected in these azole-treated cells. These results were in complete agreement with the proposed mechanism of action of azoles, which act through the inhibition of ergosterol biosynthesis, with resultant accumulation of lanosterol. Ketoconazole was found to be the least effective drug, as determined from a comparison of the effect of the three azoles on the sterol content of the four strains. Itraconazole showed to be the most effective drug, probably because of its high lipophilicity, which allows the drug to penetrate into fungi cells more efficiently.

Antifungal Agents↗
PubMed1989

Effect of hypertonic solutes upon the polysaccharide capsule in Cryptococcus neoformans.

The polysaccharide capsule is a characteristic virulence factor in the yeast-pathogen, Cryptococcus neoformans. Growth in hypertonic growth media results in yeast cells with visibly smaller capsules. We investigated this suppression quantitatively, using a chemical assay for cell-bound and dissolved capsular polysaccharide. Molar NaCl suppressed production of cell-bound polysaccharide by a factor of 2.5- to 5-fold. The possibility of salt-induced physico-chemical contraction of capsular gel was tested by dialysis of fixed cells from hypotonic medium against medium containing 1 M NaCl and against the original medium again, while capsular thickness, packed cell volume and cell-bound polysaccharide were followed. We detected a physical contraction of gel following dialysis against medium containing 1 M NaCl. Mutants which gave mucoid colonies on hypertonic agar were isolated. One of these gave twice as much polysaccharide as the wild type when cultivated in medium containing 1 M NaCl. The hypercapsular trait was passed through serial outcrosses to the wild type and segregated as a chromosomal gene. This mutant may represent a gene which regulates production of capsular polysaccharide.

Cryptococcus↗
PubMed2001

The capsular polysaccharides of Cryptococcus neoformans activate normal CD4(+) T cells in a dominant Th2 pattern.

Capsular components of Cryptococcus neoformans induce several deleterious effects on T cells. However, it is unknown how the capsular components act on these lymphocytes. The present study characterized cellular and molecular events involved in immunoregulation of splenic CD4(+) T cells by C. neoformans capsular polysaccharides (CPSs). The results showed that CPSs induce proliferation of normal splenic CD4(+) T cells, but not of normal CD8(+) T or B lymphocytes. Such proliferation depended on physical contact between CPSs and viable splenic adherent cells (SAC) and CD40 ligand-induced intracellular signal transduction. The absence of lymphoproliferation after fixation of SAC with paraformaldehyde has discarded the hypothesis of a superantigen-like activation. The evaluation of a cytokine pattern produced by the responding CD4(+) T lymphocytes revealed that CPSs induce a dominant Th2 pattern, with high levels of IL-4 and IL-10 production and undetectable inflammatory cytokines, such as TNF-alpha and IFN-gamma. Blockade of CD40 ligand by relevant mAb down-regulated the CPS-induced anti-inflammatory cytokine production and abolished the enhancement of fungus growth in cocultures of SAC and CD4(+) T lymphocytes. Our findings suggest that CPSs induce proliferation and differentiation of normal CD4(+) T cells into a Th2 phenotype, which could favor parasite growth and thus important deleterious effects to the host.

Animals↗
PubMed2003

Regulation of cytochrome c oxidase subunit 1 (COX1) expression in Cryptococcus neoformans by temperature and host environment.

In the study of differential gene expression of Cryptococcus neoformans, a transcript of COX1 (cytochrome oxidase c subunit 1) was identified in a serotype A strain. The transcript was upregulated at 37 degrees C compared to 30 degrees C and expressed by yeasts infecting the central nervous system. Northern analysis of COX1 from the serotype A strain revealed two polycistronic transcripts, a temperature-upregulated 2.3 kb transcript and a 1.9 kb transcript that was not affected by temperature. In contrast, COX1 in a serotype D strain showed only a 1.9 kb polycistronic transcript plus a 1.6 kb monocistronic message, and temperature had no effect on the transcripts. The sequence of COX1 revealed similar coding regions between the two strains, but the serotype D strain had five introns whereas no introns were found in the serotype A strain. The serotype D strain had reduced growth rates compared to the serotype A strain at 37 degrees C, but in an AD hybrid strain the serotype D COX1 gene could support efficient high temperature growth. These studies have revealed mitochondrial molecular differences between serotype A and D strains which show evolutionary divergence. It will be important to determine whether differences in mitochondrial structure and function can influence cryptococcosis.

Cryptococcus neoformans↗
PubMed2002

Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function.

Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4-5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites-acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and gamma-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas.

Animals↗
PubMed1975

Phagocytosis of Cryptococcus neoformans by alveolar macrophages.

Guinea pig pulmonary macrophages phagocytized but did not kill nonencapsulated cells of Cryptococcus neoformans. The phagocytic process was inhibited by cryptococcal capsular polysaccharide. Pulmonary macrophages, activated by preinjecting heat-killed bacteria into intact animals, did not kill the engulfed yeast cells. Labeled cells of C. neoformans were neither killed nor cleared from guinea pig lungs 6 h postexposure. The results of our experiments indicate that during the first few hours after the lung is exposed to the infectious particle of C. neoformans the pulmonary macrophage does not function primarily to kill engulfed yeast cells. We believe that a rapid yet transient acute inflammatory response probably plays a major role in this process during the first few hours after C. neoformans enters the lung.

Acid Phosphatase↗
PubMed1999

Antifungal activity of cerebrospinal fluid against Cryptococcus neoformans and Candida species.

The effect of human cerebrospinal fluid (CSF) on the growth of Cryptococcus neoformans and Candida species was tested in RPMI-1640. CSF alone was highly fungistatic for both yeasts and inhibited growth in a concentration-dependent manner. Unlike human serum, CSF did not collaborate with fluconazole for killing C. neoformans. Molecular sieve fractionation of CSF on a G-200 Sephadex column yielded a highly antifungal fraction with a molecular weight around 66 kDa. On SDS-PAGE this fraction migrated as a major and a minor band corresponding to the mobility of bovine serum albumin. These novel findings suggest that CSF contains a factor(s) that provides resistance to the growth of C. neoformans or Candida species.

Animals↗
PubMed1998

The different binding patterns of two immunoglobulin M monoclonal antibodies to Cryptococcus neoformans serotype A and D strains correlate with serotype classification and differences in functional assays.

Cryptococcus neoformans var. neoformans strains have historically been divided into serotypes A and D on the basis of reactivity with rabbit sera. Previously, we noted that two murine immunoglobulin M monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan produced different indirect immunofluorescence (IF) patterns, described as annular and punctate, when bound to C. neoformans cells from different strains. In this study, we examined the reactivity of these two MAbs, known as 12A1 and 13F1, with 20 C. neoformans var. neoformans strains, of which 13 were serotype A and 7 were serotype D. For all strains, MAb binding was studied by IF and agglutination assays. In addition, we blindly tested the IF patterns of 22 C. neoformans var. neoformans strains. For selected strains, MAb binding was studied by flow cytometry (FACScan) and phagocytosis assays. The epitopes recognized by MAbs 12A1 and 13F1 were found in all of the strains. MAb 12A1 binding produced an annular IF pattern with all of the strains, irrespective of the serotype classification. MAb 13F1 binding produced annular binding with all of the serotype A strains and punctate binding with 19 of 20 serotype D strains. In general, the punctate IF pattern was associated with lower fluorescence intensity, a requirement for higher antibody concentrations to produce yeast cell agglutination, and lower opsonic efficacy. Our results provide strong support for the existing classification of two serological types for strains assigned to variety neoformans and indicate qualitative and quantitative antigenic differences among serotype A and D strains.

Animals↗
PubMed1993

Ultrastructural study of Cryptococcus neoformans by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of Cryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method. C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10(7) cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells of C. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure of C. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10-13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions by C. neoformans.

Animals↗