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PubMed2000

Prevalence of Cryptococcus neoformans var. neoformans (Serotype D) and Cryptococcus neoformans var. grubii (Serotype A) isolates in New York City.

Analysis of 40 New York City Cryptococcus neoformans isolates revealed that 39 were typeable, of which 85 and 12.5% were Cryptococcus neoformans var. grubii (serotype A) and Cryptococcus neoformans var. neoformans (serotype D), respectively. The prevalence of serotype D isolates in New York City appears to be significantly higher than indicated by previous studies of North American isolates.

Cryptococcus neoformans↗
PubMed2004

Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS.

Cryptococcus flavescens, a strain originally identified as C. laurentii, was isolated from the cerebrospinal fluid of an AIDS patient, and the soluble capsular polysaccharide of the yeast was investigated. Glucuronoxylomannan (GXM) was obtained from C. flavescens under conditions similar to those used to obtain C. neoformans polysaccharide. However, the GXM differed from C. neoformans polysaccharide in the decreased O-acetyl group content. The structure of GXM was determined by methylation analysis, partial acid hydrolysis, NMR analyses, and controlled Smith degradation. These analyses indicated that GXM has the following structure: an alpha-(1-->3)-D-mannan backbone with side chains of beta-D-glucuronic acid residues bound to the C-2 position of the mannose residue. The C-6 position of the mannose is substituted with D-man-beta-(1-->4)-D-xyl-beta-(1--> disaccharide. Furthermore, the existence of side chains containing more than two xylose residues was suggested. This mannosylxylose side chain is a novel structure in polysaccharides of C. neoformans and other Cryptococcus species.

Acquired Immunodeficiency Syndrome↗
PubMed2005

High frequency transformation of Cryptococcus neoformans and Cryptococcus gattii by Agrobacterium tumefaciens.

Cryptococcus neoformans and Cryptococcus gattii are the caus-ative agents of cryptococcal meningoencephalitis and are amenable to genetic manipulations, making them important models of pathogenic fungi. To improve the efficiency of Agrobacterium tumefaciens mediated transformation (ATMT) in C. neoformans, we optimized various co-cultivation conditions including incubation time and temperature, and bacteria to yeast ratio. ATMT was also applied to both serotypes (B and C) of C. gattii. Transformation efficiency by ATMT in C. neoformans was comparable to either electroporation or biolistic transformation and gave superior efficiencies in serotypes B and C, but unlike Saccharomyces cerevisiae, adenine auxotrophy did not increase ATMT efficiency in C. neoformans or C. gattii. All transformants tested were stable, with a majority containing only a single T-DNA insertion; however, homologous recombination was not observed. Additionally, we isolated adenine auxotrophs containing a single T-DNA insertion in the ADE2 gene for representative serotype B and C strains.

Agrobacterium tumefaciens↗
PubMed2006

Looking for sex in the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii.

Why are we interested in understanding the mode of reproduction being used by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii? Empirical evidence has finally supported the long-held assumption that, by increasing the rate of adaptive evolution, sex increases the chances of long-term survival. Understanding the ability of pathogenic organisms to adapt to diagnostic and treatment regimes is also important in the fight against the diseases caused by these organisms. This review looks at the different approaches used to identify population structure in C. neoformans and C. gattii. These are sexual species; however, recombination in natural populations has only recently been found. We highlight the importance of population selection and the value of both indirect molecular analysis and direct biological evidence for sexual recombination, when looking for the mode of reproduction in these fungal pathogens.

Cryptococcosis↗
PubMed2006

Cryptococcus neoformans and Cryptococcus gattii isolated from the excreta of psittaciformes in a southern Brazilian zoological garden.

Cryptococcus neoformans, a major pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from soils, avian excreta and plant material. To further study potential saprophytic sources of this yeast in the Southern Brazilian State Rio Grande do Sul, we analyzed fecal samples from 59 species of captive birds kept in cages at a local Zoological Garden, belonging to 12 different orders. Thirty-eight environmental isolates of C. neoformans were obtained only from Psittaciformes (Psittacidae, Cacatuidae and Psittacula). Their variety and serotype were determined, and the genetic structure of the isolates was analyzed by use of the simple repetitive microsatellite specific primer M13 and the minisatellite specific primer (GACA)(4) as single primers in the PCR. The varieties were confirmed by pulsed-field gel electrophoresis (PFGE). Thirty-three isolates (87%) were from the var. grubii, serotype A, molecular type VNI and five (13%) were Cryptococcus gattii, serotype B, molecular type VGI. All the isolates were mating type alpha. Isolates were screened for some potential virulence factors. Quantitative urease production by the environmental isolates belonging to the C. gattii was similar to the values usually obtained for clinical ones.

Animals↗
PubMed1998

Effects of Cryptococcus humicola killer toxin upon Cryptococcus terreus envelope: combined fluorometric and microscopic studies.

Killer toxin (microcin) produced by Cryptococcus humicola 9-6 induced interaction of the fluorogenic dyes, ethidium bromide, propidium iodide, and hemimagnesium 8-anilino-1-naphtalenesulfonate, with the sensitive strain of Cryptococcus terreus VKM Y-2253. The toxin also made the cells susceptible to cetyltrimethylammonium bromide and leaky for K+. When excited at 360 nm, cell-bound ethidium (propidium) fluorescence was enhanced by 8-anilino-1-naphtalensulfonate, and cell-bound 8-anilino-1-naphtalensulfonate fluorescence was quenched by ethidium (propidium), indicating energy transfer from 8-anilino-1-naphtalensulfonate to ethidium (propidium). These results suggest that at least a portion of the probe molecules had the same binding site, possibly the cytoplasmic membrane. The parameters of kinetics of microcin action were evaluated fluorometrically. They were found to be identical for all probes and depended on microcin concentration. The fluorescence increment of ethidium and 8-anilino-1-naphtalensulfonate upon binding to microcin-treated cells correlated with the fraction of stainable cells and viability.

Bacteriocins↗
PubMed2005

Isolation of Cryptococcus gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India.

The association of Cryptococcus gattii with Eucalyptus trees has been well established. Here we report the isolation of both C. gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. We investigated a total of 233 samples of Eucalyptus trees: 120 flowers, 81 fragments of bark, and 32 leaves. C. gattii was isolated from two samples of flowers of Eucalyptus terreticornis. C. neoformans var. grubii was recovered twice from the bark of Eucalyptus camaldulensis, initially from one of three samples, and again 2 months later, from one of four samples collected beneath the canopy of the tree. The primary isolation medium was Nigerseed agar, and brown colonies were presumptively identified as C. gattii or C. neoformans. The species identification was confirmed by morphological and biochemical characteristics. Using the Crypto-Check kit (Iatron, Tokyo, Japan), the first two isolates were identified as serotype B (C. gattii) and the other two were serotype A (C. neoformans var. grubii). PCR analysis of the isolates of C. neoformans var. grubii revealed that they possessed the MATalpha mating type allele. Molecular typing by amplified fragment length polymorphism markers indicated that both isolates of C. neoformans var. grubii possessed the same genotype. This study demonstrates that C. neoformans var. grubii, as well as C. gattii, may be associated with Eucalyptus trees.

Cryptococcus neoformans↗
PubMed2006

Experimental systemic infection with Cryptococcus neoformans var. grubii and Cryptococcus gattii in normal and immunodeficient mice.

Cryptococcus neoformans (Cn) var. grubii or Cryptococcus neoformans var. neoformans infection is usually associated with immunocompromised hosts, whereas Cryptococcusgattii more frequently causes disease in immunocompetent hosts. We examined the effects of immunodeficiency and glucocorticoid-induced immunosuppression on systemic murine infection induced by i.v. inoculation with these pathogens. SCID and immunocompetent BALB/c and C57BL/6 mice were infected with <or=107 yeast of Cn var. grubii or C. gattii; immunosuppressed BALB/c mice were infected with <or=106 yeast. Mortality was inoculum size-dependent in each model system, for both organisms. Following infection with 106 CFU of either Cn var. grubii or C. gattii immunocompetent BALB/c mice survived longer than immunosuppressed mice (P<0.0001 in both cases); no differences were found using lower inocula. SCID mice infected with Cn var. grubii or C. gattii died sooner than BALB/c mice (P<0.0013, all comparisons). Unexpectedly, BALB/c mice infected with C. gattii developed external lesions. Immunocompetent mice developed rectal prolapse more frequently whereas immunosuppressed mice developed more frequent skin lesions, predominantly on the tail. The fungal burden was especially high in rectum, skin and lung tissues. Histologic examination showed extensive infection of the rectum and skin and pneumonitis. Determination of CFU from various organs of immunocompetent BALB/c mice infected i.v. with 105 CFU of C. gattii or Cn var. grubii showed significant temporal increase of burdens of Cn var. grubii in brain and liver (P<0.003); other organs showed decreasing fungal burden. C.gattii was recovered only from liver and lungs, no CFU were detected in the other organs. As opposed to epidemiologic observations, our results demonstrate no predilection by C. gattii for infection of immunocompetent over immunosuppressed hosts; immunosuppression increased the risk of severe cryptococcosis by both varieties, especially at high inocula. This is the first report of C. gattii inducing experimental cutaneous and intestinal mucosal infection; Cn var. grubii did not affect these tissues, indicating differences in tissue tropism of these pathogens.

Animals↗
PubMed2006

Emergence of amphotericin B-resistant Cryptococcus laurentii meningoencephalitis shortly after treatment for Cryptococcus neoformans meningitis in a patient with AIDS.

A unique occurrence of two subsequent episodes of HIV-associated Cryptococcus neoformans followed by C. laurentii meningoencephalitis (successfully cured with fluconazole after demonstrated amphotericin B resistance) is presented and discussed. The available literature reported only two cases of HIV-associated C. laurentii infection to date, while a concurrent infection by C. neoformans and C. laurentii has been reported only once in a patient without HIV disease. The absence of prior descriptions of documented central nervous system infection by C. laurentii in non-HIV-infected patients is emphasized as well as the unpredictable in vitro and in vivo antifungal susceptibility of this non-neoformans Cryptococcus spp., and its apparent prevalence in hospitalized patients who received prior antifungal treatments. All epidemiologic, diagnostic, clinical, and therapeutic implications are discussed on the basis of the evolving characteristics of opportunism in the era of highly active antiretroviral therapy (HAART).

AIDS-Related Opportunistic Infections↗
PubMed2002

Cryptococcus statzelliae sp. nov. and three novel strains of Cryptococcus victoriae, yeasts isolated from Antarctic soils.

A morphological and physiological characterization of yeast strains CBS 8908, CBS 8915, CBS 8920, CBS 8925(T) and CBS 8926, isolated from Antarctic soils, was performed. Phylogenetic analyses of the sequences of the D1/D2 regions and the adjacent internal transcribed spacer (ITS) regions of the large-subunit rDNA of these strains placed them into the Tremellales clade of the Hymenomycetes. The sequence data identified strains CBS 8908, CBS 8915 and CBS 8920 as belonging to the species Cryptococcus victoriae. Strains CBS 8925(T) and CBS 8926 were found to represent an unique clade within the Hymenomycetes, with Dioszegia crocea CBS 6714(T) being their closest phylogenetic relative. Fatty acid composition and proteome fingerprint data for these novel strains were also obtained. No sexual state was observed. A novel basidiomycetous species, Cryptococcus statzelliae, is proposed for strains CBS 8925(T) and CBS 8926.

Antarctic Regions↗
PubMed1997

Biochemical comparison of the Cu,Zn superoxide dismutases of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii.

Cu,Zn superoxide dismutases (SODs) have been purified to homogeneity from the two varieties of Cryptococcus neoformans, C. neoformans var. neoformans and var. gattii. The N-terminal amino acid sequences of the two enzymes were similar, though not identical, and demonstrated homology with Cu,Zn SODs from other organisms. SOD activity was present in supernatants from stationary-phase cultures of isolates of C. neoformans var. neoformans and was also present from the mid-log phase onwards in cultures of an acapsular mutant of C. neoformans var. neoformans. SOD activity was practically undetectable in culture supernatants from isolates of C. neoformans var. gattii. The C. neoformans var. neoformans SOD had a reduced relative molecular mass of 19 kDa, and in its nonreduced form the enzyme was present as a 125-kDa species. Isoelectric focusing indicated that four species with pIs of 5.9, 6.15, 6.35, and 6.6 were present. The equivalent reduced molecular mass of the C. neoformans var. gattii enzyme was 19 kDa, with a single species present under nonreducing conditions (relative molecular mass of 145 kDa) with a pI of 7.5. The activities of the enzymes from both varieties were inhibited by KCN; however, the copper chelator diethyldithiocarbamate was inhibitory only against the C. neoformans var. gattii enzyme, as was sodium azide. The C. neoformans var. neoformans SOD was not affected by preincubation for 1 h at 70 degrees C, and it also retained most of its activity when incubated at 37 degrees C relative to its activity when incubated at 20 degrees C, in contrast to the C. neoformans var. gattii enzyme. The pronounced differences in the physical and biochemical characteristics of the Cu,Zn SODs from the two Cryptococcus varieties complement recent reports illustrating the biochemical and genetic differences between C. neoformans var. neoformans and C. neoformans var. gattii, and the successful purification of the two enzymes comprises the first step in determining what role, if any, the cryptococcal Cu,Zn SODs might have in protection against externally generated superoxide.

Amino Acid Sequence↗
PubMed1998

First report of Cryptococcus laurentii meningitis and a fatal case of Cryptococcus albidus cryptococcaemia in AIDS patients.

We report the first case of Cryptococcus laurentii meningitis and a rare case of Cryptococcus albidus cryptococcaemia in AIDS patients. Both infections were treated with amphotericin B and flucytosine. The C. laurentii meningitis was controlled after 2 weeks of treatment with no evidence of infection 20 months later. The patient with C. albidus cryptococcaemia, despite the amphotericin B/flucytosine combination therapy, died on the 14th day of treatment. The minimum inhibitory concentrations (MICs) for C. laurentii, as determined by Etest on RPMI 1640 agar, were 0.25 microg ml(-1) of amphotericin B, 1.25 microg ml(-1) flucytosine, 4 microg ml(-1) fluconazole, 0.50 microg ml(-1) itraconazole and 1.0 microg ml(-1) of ketoconazole. The MIC of amphotericin B for C. albidus was 0.5 microg ml(-1), flucytosine 1.25 microg ml(-1), fluzonazole 4 microg ml(-1), itraconazole 0.5 microg ml(-1) and ketonazole 0.25 microg ml(-1). The agreement of the amphotericin B MIC values obtained in antibiotic medium 3 by the broth microdilution method, with those obtained on casitone medium by Etest, was within a two-dilution range for both isolates. C. laurentii may cause meningitis and may also involve the lungs in AIDS patients.

AIDS-Related Opportunistic Infections↗
PubMed1986

Comparative study of the temperature profiles of growth and death of the pathogenic yeast Cryptococcus neoformans and the non-pathogenic Cryptococcus albidus.

The temperature profiles of two species of Cryptococcus were compared. The pathogenic Cr. neoformans had a maximum temperature for growth of 39.8 degrees C and the non-pathogenic Cr. albidus, of 30.2 degrees C. The specific growth rates measured in the former were of an order of magnitude higher than in the latter, whereas the Arrhenius plots of the specific thermal death rates did not show a significant difference.

Cryptococcus↗
PubMed1991

Comparison of serological and chemical characteristics of capsular polysaccharides of Cryptococcus neoformans var. neoformans serotype A and Cryptococcus albidus var. albidus.

The antigenic formula and chemical structure of capsular polysaccharide (CPS) of Cryptococcus albidus var. albidus (C. albidus) were studied in relation to those of C. neoformans var. neoformans serotype A (C. neoformans A). The results of slide agglutination tests with factor sera and reciprocal adsorption experiments showed that antigenic formula of C. albidus was the same as that of C. neoformans A. The soluble CPSs from the two species were obtained from culture supernatants by precipitation with ethanol followed by purification by chromatography on DEAE-cellulose column. The structural analyses of such CPSs from the two species showed that the antigenic CPS fractions consisted of a backbone of alpha(1-3)-linked D-mannopyranosyl residues with a single branch of beta(1-2)-xylose or glucuronic acid, and mostly with O-acetyl groups, in which side chains and O-acetyl groups were responsible for antigenic specificity. It was found that there was a minor difference between the CPS of C. neoformans A and that of C. albidus; in the former, unsubstituted mannose residues existed in a low frequency, but in the latter none. Moreover, the 1H-nuclear magnetic resonance spectra of partially hydrolyzed acidic fragments of the two CPSs indicated that two xylose side chains were present between glucuronic acid side chains. Taken together, it was suggested that these two species of C. neoformans A and C. albidus are closely related to each other in their CPSs.

Animals↗
PubMed1980

Creatinine metabolism in Cryptococcus neoformans and Cryptococcus bacillisporus.

The pathogenic species of Cryptococcus, C. neoformans and C. bacillisporus, utilized creatinine as a source of nitrogen but not of carbon. Chromatographic and autoradiographic studies suggest that creatinine metabolism in both species involves a single step resulting in the production of methylhydantoin and ammonia. The enzyme responsible for this step, creatinine deiminase, was produced by the cells only in the presence of creatinine in both species. The synthesis of creatinine deiminase was repressed by ammonia in C. neoformans, but not in C. bacillisporus. A possible explanation for this variation, based on the ecological differences between the two species, is discussed. A novel method for measuring creatinine deiminase activity is also described.

Aminohydrolases↗
PubMed1982

Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C).

A simple new agar medium containing L-canavanine, glycine, and bromthymol blue was found to give a clearer and more accurate distinction between serotype A or D (Cryptococcus neoformans var. neoformans) and serotype B or C (C. neoformans var. gattii) than creatinine-dextrose-bromthymol blue or glycine-cycloheximide-phenol red media. Among 143 isolates of serotype A or D and 70 isolates of serotype B or C, the new medium correlated completely with the serotype, whereas nearly 11% of these isolates gave discrepant reactions with creatinine-dextrose-bromthymol blue and glycine-cycloheximide-phenol red media.

Cryptococcus↗
PubMed1979

Further simplification of the Guizotia abyssinica seed medium for identification of Cryptococcus neoformans and Cryptococcus bacillispora.

A simplified Guizotia abyssinica seed-based medium for presumptive diagnosis of Cryptococcus neoformans and C. bacillispora (Paliwal and Randhawa 1978) was further simplified by replacing seed extract with pulverized seeds. This medium gives unambiguous results, avoids false-positive reactions with 13 other yeastlike organisms, and is simple and relatively inexpensive to prepare.

Cryptococcus↗