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PubMed1984

Enzyme immunoassay detection of IgM to galactoxylomannan of Cryptococcus neoformans.

Antibodies against the major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), and a minor secreted polysaccharide, galactoxylomannan (GalXM), were surveyed by indirect enzyme immunoassay (EIA) in patients with cryptococcosis, with other mycoses, and in normal controls. Measurement of IgG levels against GalXM revealed cross reactions in candidiasis patients that were reduced by adsorption with Candida albicans cell walls. Measurement of IgM levels were subject to fewer cross reactions. The combination of adsorption with C albicans cell walls and measurement of IgM detected antibodies in 12 of 55 cryptococcosis patients. An end point equal to or greater than a titer of 1/16 excluded reactions in normals and limited cross reactivity in candidiasis patients to below 7%. This test has potential diagnostic significance in cryptococcosis patients who show no evidence of cryptococcal antigen circulating in the cerebrospinal fluid or serum. Reactions in this IgM assay were not spuriously due to rheumatoid factor. The major capsular GXM was much less serologically active and was subject to cross reactions with agents of bacterial sepsis. The specificity of the GalXM is directed mainly by the mannose and to a lesser extent by galactosyl residues.

Antibodies, Anti-Idiotypic↗
PubMed1984

Lipid composition of Cryptococcus neoformans.

The lipid composition of Cryptococcus neoformans grown in Sabouraud's dextrose broth (shake culture) was analysed. The organism contained extremely low amounts of lipid (0.96% dry weight basis) of which 86.1% were nonpolaris lipids, 3.4% phospholipids and the rest were glycolipids and pigments. Alkoxylipids (41%), tryglicerides (18%), diglycerides (7.4%), free fatty acids (5.4%), sterols (4.7%), sterol ester (3.9%) and monoglycerides (2.2%) were found in the nonpolar lipid fraction of C. neoformans. The phospholipid composition (expressed as relative abundance) was: phosphatidylinositol (11.5%), lysophosphatidyl ethanolamine (10.9%), cardiolipin (10.1%), a glycophospholipid (9.5%), lysophosphatidyl choline (4.7%), phosphatidic acid (4.1%), phosphatidyl choline (28.1%), phosphatidyl ethanolamine (14.5%) and an unidentified lipid (6.5%). Phosphatidyl serine, sphingolipids and cerebrosides, generally found in yeast-like fungi, were absent. Probable reasons for the abnormally low lipid content are discussed.

Chromatography, Thin Layer↗
PubMed1984

Virulence, capsule size and lipid composition interrelation of Cryptococcus neoformans.

Virulence and lipid composition were studied in three isolates of Cryptococcus neoformans. Virulence was evaluated by injecting mice intraperitoneally with 10(7) cells and recording organ involvement and spontaneous death over a 25 day period. Though the least virulent strain contained the least amount of total lipid and phospholipids, none of the lipids showed any quantitative relation to virulence. There was no major difference in the phospholipid composition among the three strains. Fungal cells with bigger capsules had a lower lipid content. The role of lipid in the defence mechanism of pathogenic fungi during the host invasion process is discussed.

Animals↗
PubMed1982

Isolation of Cryptococcus neoformans from pigeon manure on two media inducing pigment formation.

A newly described medium with esculin for identification of Cryptococcus neoformans was compared with Staib's Guizotia abyssinica extract-creatinine medium (GAEC) with and without diphenyl (DF). Twenty-seven samples of pigeon manure were examined. Cr. neoformans was found in 6 samples (22%) on GAEC plates (-DF); ESC medium (-DF) and malt extract agar allowed isolation from 2 and 3 samples respectively. Cr. neoformans was found in 0 to 2 samples when DF was added. Colonies of Cr. neoformans found on ESC plates had no distinctive pigmentation although inocula of pure cultures produced brown colonies. On GAEC plates some colonies of Cr. neoformans turned brown not until after 2 weeks of incubation. At 1 month the presence of pigmented colonies on GAEC plates (-DF) allowed the identification of 5 of the 6 samples from which Cr. neoformans was isolated. Other yeasts were grown from 26 samples (96%) and Torulopsis candida was found to be more frequent than Cr. neoformans.

Agar↗
PubMed1982

Control of Cryptococcus neoformans in nature by biotic factors.

Two bacterial species (Pseudomonas aeruginosa and Bacillus subtilis) isolated from pigeon droppings, displayed anti Cryptococcus neoformans activity on 4 of 6 media and sterilized pigeon droppings. Acanthamoeba palestinensis trophozoites isolated from pigeon droppings ingested and killed 99.9% of C neoformans cells after 7 days of incubation. Mites and sow bugs (Metoponorthus pruinosus) isolated from pigeon droppings appear to be fungivorous. These findings suggest that many organisms that occur in pigeon droppings influence C. neoformans persistence, reproduction, morphology and distribution in nature.

Amoeba↗
PubMed1980

Auxanographic detection of experimental murine uremia with Cryptococcus neoformans.

A modified blood residual nitrogen plate auxanographic method was applied to the detection of experimental uremia in a murine model. The yeast-like fungus, Cryptococcus neoformans, was used as the indicator. Transient uremia was induced by injection of 0.2 ml glycerol intramuscularly. The low molecular weight nitrogen levels were estimated by measuring the diameter of the auxanogram at intervals of 2 hr for 24 hr and at 32 hr after the glycerol injection. After 4 hr, elevated levels of low molecular weight nitrogen were found. Maximum levels occurred 20 hr post glycerol injection. This method requires only 5 microliter of whole blood per assay. The results can be read after an incubation time of 24 hr at 26 degree C. The stability of the prepared plates was determined to be at least 96 hr at 4 degree C. The ease of use, reliability and versatility of the modified auxanographic method are discussed.

Animals↗
PubMed1981

The perfect state of Cryptococcus neoformans, Filobasidiella neoformans, on pigeon manure filtrate agar.

To enable studies of the dependence of Cryptococcus neoformans and its perfect and imperfect states upon bird manure as a habitat of this pathogen, a nutrient medium closely resembling natural conditions was prepared. As sole nutrient, the water soluble ingredients of manure from pigeons (Columbia livia) were used. There was no heat sterilization of the manure filtrate. Using a standard pair of C. neoformans strains for mating, it could be demonstrated that the perfect state of the fungus developed on this so called pigeon manure filtrate agar within 48 h at 26 degrees C. This medium is supposed to help in the elucidation of the epidemiological significance of the perfect and imperfect states of this pathogen.

Agar↗
PubMed1982

Comparative ocular pathogenicity of Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus in the rabbit.

In a previous study, 88% of rabbits with disseminated infection caused by Candida albicans developed ophthalmoscopically visible, hematogenous endophthalmitis (chorioretinitis) over a 2 week period. To determine the incidence of this ocular complication in disseminated infection caused by Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus compared with that caused by C. albicans, the first three species of fungi were injected intravenously (between 10(5) and 10(9) organisms per animal) into 36 New Zealand white rabbits. No chorioretinal lesions were seen by indirect ophthalmoscopy over a 2 week period. C. glabrata and A. fumigatus were not cultured from chorioretinas despite positive cultures from brains and kidneys at 1 and 2 weeks. In contrast, C. neoformans was cultured from 12 of 18 chorioretinas. (mean Log10 3.45 colony forming units/gm of tissue) as well as from the brains and kidneys. The less intense inflammatory cell response to C. neoformans compared with that 10 C. albicans seen on histopathologic examination most likely explains the nondetectability of the cryptococcal chorioretinitis by indirect ophthalmoscopy. These data suggest that C. glabrata. A. fumigatus, and possibly C. neoformans have less ocular pathogenicity than C. albicans in rabbits and correlate with the small number of documented human cases of ophthalmoscopically visible hematogenous endophthalmitis caused by fungi other than C. albicans.

Animals↗
PubMed1982

Combined in vitro activity of amphotericin B and 5-fluorocytosine against Cryptococcus neoformans and Candida albicans.

The in vitro combined antifungal activity of amphotericin B (AMB) and 5-fluorocytosine (5-FC) against 10 strains of Candida albicans and 8 strains of Cryptococcus neoformans was studied. Serial twofold dilutions of AMB (0.1 to 12.8 micrograms/ml) were tested with serial twofold dilutions of 5-FC (0.39 to 400 micrograms/ml) by using the broth dilution method. A synergistic effect was seen for 17 strains with 50 micrograms of 5-FC per ml added to AMB. Only one strain was killed by 0.8 micrograms per ml or less of AMB alone, whereas all strains were killed with the addition of 50 micrograms of 5-FC per ml.

Amphotericin B↗
PubMed1982

The capsule of cryptococcus neoformans passively inhibits phagocytosis of the yeast by macrophages.

We examined the mechanism by which cryptococcal capsular polysaccharide inhibits phagocytosis of Cryptococcus neoformans by macrophages. O-Acetyl and carboxyl groups are major structural features of serotype D polysaccharide. Serotype D capsular polysaccharide or encapsulated whole cryptococci were de-O-acetylated by alkaline hydrolysis with 0.1 M NaOH. Carboxyl groups were reduced by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide followed by reduction with sodium borohydride. De-O-acetylated or carboxyl-reduced polysaccharides had phagocytosis-inhibiting properties that did not differ appreciably from the untreated polysaccharide; thus, neither the O-acetyl nor the carboxyl groups were essential phagocytosis-inhibiting determinants. Antiserum specific for these noninhibitory groups was obtained by adsorption of cryptococcal antiserum with de-O-acetylated or carboxyl-reduced whole cells to produce antiserum enriched respectively with antibody specific for the O-acetyl and carboxyl groups. These adsorbed antisera showed opsonic activity for the untreated yeast that was similar to the unadsorbed antiserum when these antisera were compared at identical levels of precipitating antibody. We also examined the ability of Fab' fragments of opsonic IgG to reduce the amount of anticapsular IgG needed to opsonize the yeast. No synergy was noted between Fab' fragments and undigested IgG in opsonization. These results are consistent with a passive mechanism for inhibition of phagocytosis. The capsule does not directly modulate phagocytic function, but instead, presents a surface that is not recognized by the phagocyte. This absence of recognition is corrected by opsonizing antibody specific for any surface determinant on the capsule.

Animals↗
PubMed1975

Cryptococcus neoformans of saprophytic origin.

Cryptococcus neoformans was isolated on 276 occasions as follows: from pigeon faeces, 159 (45%) of 349 samples; pigeon crops, 39 (50%) of 80 crops; from drinking water, 37 (74%) of 50 samples; from the atmosphere, 3 strains in a contaminated loft. It was also recovered from 38 (19%) of 200 samples of canary faeces. It is possible that C. neoformans survives in the crop of pigeons and occasionally passes to the faeces where it multiplies. The factors influencing its presence are: the irregular cleaning of lofts, overcrowding and air-pollution from poor ventilation.

Air Microbiology↗
PubMed1976

Immunization of mice with a mutant of Cryptococcus neoformans. Characterization of the mutant, actively acquired resistance to experimental cryptococcosis in mice.

In 69% of subdermally and 50% of subcutaneously immunized mice, immunity against Cryptococcus neoformans was obtained. The vaccine consisted of a gamma-irradiated mutant, which grew well at 5 degree C and at room temperature. Many subcultures and animal passages did not restore this mutant to the encapsulated, pathogenic wild type.

Animals↗
PubMed1976

Caffeic acid-containing medium for identification of Cryptococcus neoformans.

A new growth medium containing caffeic acid and ferric citrate is described. The pigment produced on this medium is specific for the identification of Cryptococcus neoformans and differentiates it from other cryptococci. The medium is more easily compounded and requires less time for pigment formation than the conventional Guizotia extract media. The medium is stable in the dry form as well as in the prepared form.

Caffeic Acids↗
PubMed1976

Growth of Cryptococcus neoformans on uric acid agar.

Effect of certain low molecular nitrogen substances, namely uric acid, eura and creatinine as sole source of nitrogen was studied on 31 strains of Cryptococcus neoformans as well as on a rough looking isolate recovered from the brain of a mouse inoculated with a mucoid strain of C. neoformans. Uric acid as a nitrogen source caused striking alterations in the morphology of C. neoformans. In view of the facts that uric acid is a common end-product of human and animal metabolism, it is abundantly present in the avian faecal matter and is capable of inducing mucoid growth and capsule formation in dry growing non-encapsulated strains or in an otherwise rough looking hypha forming isolate, its role in studying the phylogenesis of C. neoformans and its pathogenicity seems to be an important proposition.

Animals↗
PubMed1979

Immunoperoxidase localization of Sporothrix schenckii and Cryptococcus neoformans. Staining of tissue sections fixed in 4% formaldehyde solution and embedded in paraffin.

An indirect immunoperoxidase staining method has been successfully applied to 4% formaldehyde solution tissue sections fixed in and embedded in paraffin for the localization of Sporothrix schenckil and Cryptococcus neoformans without prior trypsinization of tissue sections. A comparison of this method with an analogous immunofluorescence staining technique has been made.

Cryptococcus↗
PubMed1996

Isolation and characterization of a delta-9 fatty acid desaturase gene from the oleaginous yeast Cryptococcus curvatus CBS 570.

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the delta-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned delta-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the delta-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.

Amino Acid Sequence↗
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↗