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PubMed1989

Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins.

Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with cryptococci opsonized with the reconstituted pathway compared with that observed when yeast cells were opsonized with normal serum.

Complement Activation↗
PubMed1989

Murine natural killer cell interactions with a fungal target, Cryptococcus neoformans.

Earlier investigations have shown that murine natural killer (NK) cells bind to and inhibit the growth of the fungal pathogen Cryptococcus neoformans in vitro and in vivo. To define the stages of NK cell-mediated inhibition of C. neoformans growth and the requirements for the completion of these stages, the events which lead to cryptococcal growth inhibition were compared with those previously elucidated for NK cell-mediated tumor cell lysis. Our data indicate that NK cell-cryptococci binding is a distinct event that precedes inhibition; is temperature independent, although it is slowed at 4 degrees C; and is Mg2+ dependent. In contrast to binding, NK cell-mediated cryptococcal growth inhibition is temperature, Mg2+, and Ca2+ dependent. The removal of Ca2+ by EDTA addition within 3 h after maximal NK cell-cryptococci binding significantly reduced cryptococcal growth inhibition, indicating that Ca2+ is required either late in the NK cell trigger stage or early in the inhibitory stage. These stages and requirements are similar to those previously demonstrated for the model of NK cell-mediated tumor cell lysis; however, the NK cell-cryptococci interactions are somewhat slower than the interactions which culminate in the lysis of the YAC-1 tumor cell targets. These results suggest that C. neoformans cells, although structurally distinct from the standard tumor cell targets, are capable of similar cell-to-cell interactions with NK effector cells as the tumor cell targets.

Animals↗
PubMed1989

Separation of chromosomes of Cryptococcus neoformans by pulsed field gel electrophoresis.

Chromosomes from Cryptococcus neoformans, an encapsulated yeast pathogen, were separated by contour-clamped homogeneous field gel electrophoresis. Seven strains representing all four serotypes were studied. It was found that each strain had a unique, reproducible pattern of chromosome bands which could potentially be used for strain polymorphism studies. There were between 10 and 12 chromosomes in the strains studied, with an approximate genomic size of 15,000 to 17,000 kilobases. Chromosome separation also could be used to assign locations for cloned genes, and the ribosomal DNA genes were found on one of the larger C. neoformans chromosomes. The technique of electrophoretic karyotyping should be helpful for genetic and molecular investigations into the biology of C. neoformans.

Candida albicans↗
PubMed1990

Human natural killer cells do not inhibit growth of Cryptococcus neoformans in the absence of antibody.

The interaction between human natural killer (NK) cells and yeast cells of Cryptococcus neoformans was investigated because experiments in mice indicated that NK cells inhibited the growth of C. neoformans. Strains of C. neoformans serotype A that differed in both resistance to alveolar macrophages and the size and composition of their capsules were evaluated. Human NK cells, which were isolated from normal peripheral blood, were activated by preincubation with interleukin-2 and alpha interferon to generate lymphokine-activated killer (LAK) cells. Yeast cells of C. neoformans were incubated with effector cells (NK or LAK cells); and inhibition of yeast cell growth was measured at 4, 8, and 24 h by comparing quantitative plate counts with controls consisting of yeasts in the absence of effector cells. The cytolytic activity of effector cells against target cells was confirmed by the release of radiolabel from 51Cr-labeled K-562 tumor cells. Neither NK nor LAK cells inhibited the growth of 13 strains of C. neoformans at effector to target cell ratios of as high as 500:1. Monocytes, which were isolated from the same populations of leukocytes as the NK cells, inhibited the growth of two strains of C. neoformans at effector to target cell ratios of 100:1 (92 and 46% inhibition), 50:1 (87 and 17%), and 1:1 (49 and 0%). NK cells could inhibit the growth of C. neoformans by an antibody-dependent cellular cytotoxicity mechanism in the presence of rabbit anticryptococcal antiserum at dilutions up to 1:4,000. Purified capsular polysaccharide of C. neoformans had no effect on the viability or tumoricidal activity of NK or LAK cells. These data suggest that human NK and LAK cells are not impaired by C. neoformans, and in the absence of antibody, which is rarely detectable in patients, they afford much less protection against C. neoformans than monocytes do.

Animals↗
PubMed1990

Growth inhibition of Cryptococcus neoformans by cultured human monocytes: role of the capsule, opsonins, the culture surface, and cytokines.

Despite a presumed critical role of macrophages in the host response to cryptococcal infections, previous studies have failed to show growth inhibition of encapsulated Cryptococcus neoformans by human peripheral blood cultured monocyte-derived macrophages (MO-M phi). Here, we examined whether MO-M phi could be induced to inhibit growth of an encapsulated strain and an isogenic acapsular mutant strain of C. neoformans. MO-M phi were cultured in microwells, and inhibition was measured by comparing CFU at 0 and 24 h after fungal challenge. MO-M phi cultured on plastic surfaces failed to inhibit growth of the encapsulated strain, even in the presence of pooled human serum and/or anticapsular antibody. Moreover, the presence of anticapsular antibody significantly enhanced fungal growth. However, if MO-M phi were cultured on surfaces coated with fibronectin or poly-L-lysine (but not laminin or collagen) and yeast cells were opsonized with pooled human serum, then complete growth inhibition occurred. Preincubation with various concentrations of tumor necrosis factor, granulocyte macrophage colony-stimulating factor, 1,25-dihydroxycholecalciferol, or supernatants from C. neoformans-stimulated lymphocytes failed to activate macrophages for enhanced antifungal activity. The addition of gamma interferon resulted in a significant loss of growth inhibition. For the acapsular strain, complete growth inhibition was observed regardless of the choice of culture surface, opsonins, or cytokines. Fungicidal activity, as measured by a significant decrement in CFU compared with the initial inoculum, was not observed under any conditions tested. These data demonstrate that macrophages are capable of inhibiting cryptococcal growth but that this capacity is markedly influenced by the culture surface, opsonins, cytokines, and the fungal capsule.

Antibodies, Fungal↗
PubMed1990

Production of the hexitol D-mannitol by Cryptococcus neoformans in vitro and in rabbits with experimental meningitis.

We studied the ability of Cryptococcus neoformans to produce the hexitol D-mannitol in vitro and in rabbits with experimental meningitis. Twelve of twelve human isolates of C. neoformans produced D-mannitol in yeast nitrogen base plus 1% glucose and released D-mannitol into the medium. In a pilot study, pooled cerebrospinal fluid (CSF) from cortisone-treated rabbits given 3 x 10(7) C. neoformans H99 intracisternally contained more D-mannitol (identified by gas chromatography and enzymatically) than CSF from normal controls or cortisone-untreated rabbits with self-limited meningitis. In a second experiment, cortisone-treated rabbits given C. neoformans intracisternally had significantly higher CSF D-mannitol concentrations than controls given cortisone alone at 4, 6, and 8 days after infection. Moreover, log10 CSF D-mannitol correlated well with log10 CSF CFU (r = 0.81) and log10 CSF cryptococcal antigen titers (r = 0.78). Lastly, the initial volume of distribution and elimination half-life of D-mannitol given intracisternally to normal rabbits suggested that D-mannitol was distributed in total CSF and was removed by CSF bulk flow. Thus, C. neoformans produces D-mannitol in vitro and in vivo, and D-mannitol is a quantitative marker for experimental cryptococcal meningitis. D-Mannitol produced by C. neoformans may also contribute to brain edema and interfere with phagocyte killing by scavenging hydroxyl radicals.

Animals↗
PubMed1990

Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide.

A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.

Animals↗
PubMed1990

Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), a possible adhesion receptor for yeasts.

The role of glycosphingolipids as adhesion receptors for yeasts was examined. Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, as well as Histoplasma capsulatum and Sporotrichum schenckii (in their yeast phases), bound specifically to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), as measured by overlaying glycosphingolipid chromatograms with 125I-labeled organisms. An unsubstituted galactosyl residue was required for binding, because the yeasts did not bind to glucosylceramide (Glc beta 1-1Cer) derived from lactosylceramide by treatment with beta-galactosidase or to other neutral or acidic glycosphingolipids tested that contained internal lactosyl residues. Interestingly, the yeasts preferentially bound to the upper band of the lactosylceramide doublet in human lung and bovine erythrocytes, suggesting that the ceramide structure also affects binding. Active metabolism of the yeasts was required for binding to lactosylceramide, as binding was maximal in buffer containing glucose and was almost completely abolished in nutrient-deficient medium. C. neoformans also bound to human glioma brain cells grown in monolayers, and this binding was inhibited by liposomes containing lactosylceramide but not by liposomes containing glucosylceramide. Lactosylceramide is a major glycosphingolipid in these cells and the only one to which the yeasts bound. As lactosylceramide is widely distributed in epithelial tissues, this glycosphingolipid may be the receptor for yeast colonization and disseminated disease in humans.

Antigens, CD↗
PubMed1990

Catecholamines and virulence of Cryptococcus neoformans.

Cryptococcus neoformans was unable to utilize catecholamines (epinephrine, norepinephrine, or dopamine) as sole carbon or nitrogen sources. Therefore, catecholamines are not essential growth factors for this fungus and the brain is not a preferred nutritional niche for its growth with regard to catecholamines. To establish whether the brain is a survival niche for C. neoformans and to explain the role of phenoloxidase as a virulence factor, a wild-type strain that had phenoloxidase activity and mutants which lacked it were exposed to an epinephrine oxidative system, and the survival of both strains was tested. The oxidative system contained epinephrine as an electron donor, Fe3+ as the catalytic transition metal ion, and hydrogen peroxide as an electron acceptor. The wild-type strain was found to be resistant to this oxidative system, whereas under the same conditions the mutant strain was susceptible and its survival decreased at a rate of 4 logs per h. Damage to high-molecular-weight DNA seems to be a causative factor of cell death after exposure of the mutants to the oxidative system. These results suggest that C. neoformans may survive in the brain because of its ability to utilize catecholamines for melanogenesis and thus neutralize the harmful effects of catecholamines which are manifested in the presence of hydrogen peroxide and transition metal ions. The role of phenoloxidase in resistance to the epinephrine oxidative system is also discussed.

Catechol Oxidase↗
PubMed1972

Intracellular fate of Cryptococcus neoformans.

Human peripheral leukocytes were found to engulf and kill cells of Cryptococcus neoformans. Fewer encapsulated than nonencapsulated cells met this fate, since cryptococcal capsular polysaccharide inhibited phagocytosis. During 10 to 12 hr of incubation of nonencapsulated cells in human serum, sufficient polysaccharide was produced to inhibit phagocytosis by 50%. The polysaccharide inhibitor was found in the sera of four patients with cryptococcosis, but not on the surfaces of their leukocytes. Additional experiments indicated that serum is not essential for effective phagocytosis. However, normal human serum contains anticryptococcal activity which is not inhibited by capsular material. Preliminary findings indicate that the phagocytic index of a patient with cryptococcosis may be correlated with the severity of his disease.

Animals↗
PubMed1972

Tumor induction by Cryptococcus neoformans.

A human isolate of Cryptococcus neoformans strain CIA, which was originally obtained in 1963, produced fatal disease in mice. Postmortem examinations showed extensive central nervous system disease. After using this yeast for research for 7 years, we found that it began to produce large tumors after intraperitoneal inoculation in mice. The present study showed that the tumors consisted of large agglomerates of yeast cells, capillaries, and reticular stroma. In concomitant experiments, the same C. neoformans strain, introduced into the lungs of mice in an aerosol, produced asymptomatic infections. A definition for the term "cryptococcoma" is proposed.

Aerosols↗
PubMed1973

Relationship of some Cryptococcus neoformans hypha-forming strains to standard strains and to other species of yeasts as determined by deoxyribonucleic acid base ratios and homologies.

The taxonomic relationship between some hypha-forming Cryptococcus neoformans strains and those that morphologically fit the standard description of this species was determined on the basis of DNA base ratios and DNA-DNA duplex formation. The average guanosine plus cytosine content, as estimated from melting temperature profiles, varied between 43.0 and 45.9% for all C. neoformans strains examined. The relative homology was at least 60% for all C. neoformans strains when the labeled DNA was from either a hyphal variant or a standard strain. The nonpathogenic cryptococci, C. laurentii and C. uniguttulatus, showed less than 10% relative homology with either the hyphal or standard C. neoformans strains. Thus, hyphal and standard strains of C. neoformans were sufficiently related to be considered members of the same species.

Base Sequence↗
PubMed1973

Fine structure of Cryptococcus neoformans grown in vitro as observed by freeze-etching.

Cryptococcus neoformans grown on culture media was observed by the freeze-etching technique. In the capsule, short fibrils were seen when freezeetched. This organism was unique in the appearance of the cell wall, which showed two strata. The outer one was dense with particles of about 20 nm in diameter, whereas the inner one was sparse in particles. The appearance of the cell membrane of this organism differed distinctly depending on the culture media. When grown on glycerol medium, the cell membrane possessed, as do other yeasts, clear but somewhat longer and curved invaginations. The membrane of cells grown on nonglycerol medium exhibited, however, only a few invaginations of irregular shape. Instead, characteristically of this organism, the cell membrane showed round depressions of 40 to 200 nm in diameter which were the surface view of the paramural bodies. In cross-fractured cells, both types of paramural bodies were found. Some of them contained a single vesicle of about 50 nm in diameter. These seem to play a role in secreting the cytoplasmic vesicles. Data suggesting the existence of multivesicular bodies in the cytoplasm and multivesicular lomasomes were also obtained. Some of the baglike paramural bodies showed multilayered membrane. These are thought to be plasmalemmasomes. This organism was similar to other yeasts reported in other respects.

Cell Membrane↗
PubMed1973

Fine structure of Cryptococcus neoformans grown in vivo as observed by freeze-etching.

Cryptococcus neoformans grown in the parasitic state was observed by the freeze-etching technique and was compared with that grown on culture media. Unlike other yeasts, this organism grown in vivo is very often devoid of the "ordinary" invaginations. The membrane of the cell grown in vivo was almost free from concavity and convexity except for many round depressions which represent the surface view of paramural bodies. Some of the paramural bodies were found to be multivesicular systems. Most were spherical invaginations containing a single vesicle or its ghost remaining after secretion of the vesicles. In clear contrast to the cell grown in vitro, the in vivo cell contained a great number of vesicles in the cytoplasm. These seemed to show high-secretion activity in C. neoformans grown in the parasitic state. On transfer from in vitro to in vivo, this organism enlarged the cell wall, capsule, and cell body. The appearance of a large vacuole, accumulation of storage organelles, and the existence of rodlike structures, seemingly lipid deposits, were also noted in the cytoplasm of the cell grown in vivo. the meaning of these results as well as the mode of capsular production are discussed.

Animals↗
PubMed1976

Ultrastructure of septal complex in Filobasidiella neoformans (Cryptococcus neoformans).

Electron microscopy of Filobasidiella neoformans, the perfect state of Cryptococcus neoformans, revealed basidiomycete doliporesepta between hyphal cells and also between clamp connections and adjacent cells. The pore-occluding material was a heterogeneous flattened plate with dark margins and a lighter center, as seen in the species of Filobasidium. Representative basidiomycete parenthesomes were lacking, and endoplasmic reticulum was seen in the dolipore region.

Cell Wall↗
PubMed1976

Light microscopy of basidia, basidiospores, and nuclei in spores and hyphae of Filobasidiella neoformans (Cryptococcus neoformans).

Three hypha-forming strains of Cryptococcus neoformans were induced to form basidia and basidiospores. Light microscopy showed that basidia formed at the ends of terminal hyphal cells and were able to produce from a few to many basidiospores. The morphology of the sexual structures indicated that these strains belonged to the recently described perfect state of C. neoformans, Filobasidiella neoformans. The average dimensions of the basidiospores were 1.9 mum in width by 2.7 mum in length. Giemsa staining revealed that dikaryotic cells were formed in all three strains. Only one strain had both terminal and subterminal dikaryons, indicating functional clamp connections, whereas the two remaining strains had dikaryons restricted to the terminal cells. Basidiospores of two strains were mononucleate, and yeast cell clones derived from single basidiospores of these two strains were able to complete the sexual life cycle, thus indicating their primary homothallic nature.

Cell Nucleus↗
PubMed1982

Biochemical studies of phenoloxidase and utilization of catecholamines in Cryptococcus neoformans.

Protoplasts of Cryptococcus neoformans contain phenoloxidase as a membrane-bound enzyme. The enzyme appeared to be attached on the inner side of cytoplasmic membranes. Synthesis of the enzyme was derepressed by low levels of glucose but was not affected by the level of ammonium. Copper chelators which inhibited the phenoloxidase of other organisms did not affect cryptococcal enzymes. However, cyanide- or iron-chelating agents such as hydroximide derivates or 8-hydroxyquinoline were effective inhibitors, suggesting that cryptococcal phenoloxidase is an iron-containing enzyme. Phenoloxidase of C. neoformans catalyzed the oxidation of various diphenols via dopachrome and labile intermediates to melanin polymers. The kinetic constants (Km) of the phenoloxidase and the permease for dopamine and norepinephrine were low. The correlation between phenoloxidase and the preferential growth of C. neoformans in the host brain is discussed.

Ammonia↗