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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↗
PubMed2000

Prevalence of Cryptococcus neoformans in clinical specimens.

Cryptococcus neoformans isolated from various clinical materials in 14 cases, was identified by (1) cultivation on Sabouraud glucose agar and CHROMagar Candida, (2) microscopic examination of Indian-ink-stained preparations and (3) determination of biochemical properties (assimilation and fermentation of saccharides, assimilation of KNO3, production of urease and phenol monooxygenase). C. neoformans was determined in five specimens from paediatric patients in the intensive care unit and in nine specimens from adult patients, most frequently from liquor at meningitis (n = 3).

Adult↗
PubMed1993

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

The ultrastructure of hepatic granulomas induced by Cryptococcus (C.) neoformans was studied by a quick-freezing and deep-etching (QF-DE) method. Viable yeast cells were inoculated intravenously into rats and the livers were prepared for QF-DE replicas. Two cytoskeletal components were identified in the cytoplasm of macrophages composing the cryptococcal granulomas. These were: intermediate filaments, mainly located in the perinuclear region, and actin filaments, which were extensively decorated with myosin subfragment 1 (S1) and formed networks in the peripheral portion of the cytoplasm. In addition, two types of macrophage pseudopodia were observed in the granulomas. These were cobble stone-like pseudopodia at the yeast-macrophages contract areas, and thin, long and occasionally interdigitating pseudopodia in which actin filaments were consistently observed. Dense networks of actin filaments were also seen in pseudopodia protruding into the tight structure of the capsule of C. neoformans. These results suggest a role for actin filaments as one of the main factors in the force generating system of the phagocytic process.

Animals↗
PubMed2003

Characterization of Cryptococcus neoformans var. neoformans serotype A and A/D in samples from Egypt.

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.

Adolescent↗
PubMed2003

Influence of arsenic on antimony methylation by the aerobic yeast Cryptococcus humicolus.

The anamorphic basidomycetous yeast Cryptococcus humicolus was shown by hydride generation-gas chromatography-atomic absorption spectrometry to methylate inorganic antimony compounds to mono-, di-, and trimethylantimony species under oxic growth conditions. Methylantimony levels were positively correlated with initial substrate concentrations up to 300 mg Sb l(-1) as potassium antimony tartrate (K-Sb-tartrate). Increasing concentrations of K-Sb-tartrate increased the ratio of di- to trimethylantimony species, indicating that methylation of dimethylantimony was rate limiting. Antimony methylation capability in C. humicolus was developed after the exponential growth phase and was dependent upon protein synthesis in the early stationary phase. Inclusion of inorganic arsenic (III) or (V) species alongside antimony in culture incubations enhanced antimony methylation. Pre-incubation of cells with inorganic arsenic (III) further induced antimony methylation capability, whereas pre-incubation with inorganic antimony (III) did not. Exposure of cells to inorganic arsenic-either through pre-incubation or provision during cultivation-influenced the antimony speciation; involatile trimethylantimony species was the sole methylated antimony species detected, i.e. mono- and dimethylantimony species were not detected. Competitive inhibition of antimony methylation was observed at high arsenic loadings. These data indicate that antimony methylation is a fortuitous process, catalysed at least in part by enzymes responsible for arsenic methylation.

Aerobiosis↗
PubMed2005

Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogotá, Colombia, and study of ecological conditions in the area.

Cryptococcus neoformans, the etiological agent of cryptococcosis, has been associated with avian droppings and certain trees in different countries, including Colombia. C neoformans environmental isolates were obtained in urban areas in Bogotá, Colombia, and the strains recovered were phenotypically characterized. Attempts to determine the ecological conditions (micro- and macroclimatic) possibly related to their habitat were also undertaken. Four hundred and eighty samples from bark, soil around trunk bases, and detritus inside hollows of 32 trees were collected in three urban areas during a 5-month period, as well as 89 avian droppings samples from different places. Of plant samples, 6.7% collected from nine tree species yielded C. neoformans var. gattii, serotype B strains in 99% of the cases, and C. neoformans var. grubii, serotype A in 1%. The yeast was more frequently recovered from bark than from soil or detritus inside hollows, and from trees with hollows or rotted wood rather than from trees in which birds nest. C. neoformans was present with higher frequency and density in the rainy season than in the dry season; we found that slightly higher temperature and humidity values of the microhabitat, as compared to those of the environment, favored fungal occurrence, but the phenological state of the tree did not. Of dropping samples, 7.9% yielded C. neoformans strains, all of them C. neoformans var. grubii, serotype A. The yeast was obtained more frequently from dry droppings than from moist ones, but neither the sunlight exposure nor the site of collection of samples was correlated with this occurrence. Population density was significantly higher in droppings than in tree samples. Under laboratory conditions, isolates of different serotype showed similar capsular sizes. Water content and pH ranges were wide and did not show any significant difference between positive and negative samples.

Animals↗
PubMed1997

Cloning and expression of the delta 9 fatty acid desaturase gene from Cryptococcus curvatus ATCC 20509 containing histidine boxes and a cytochrome b5 domain.

To allow genetic modification of the fatty acid biosynthesis routes in the lipid-accumulating yeast Cryptococcus curvatus the delta 9 fatty acid desaturase gene was cloned and characterized. The 1668-bp gene encodes a protein of 556 amino acids with a calculated molecular mass of 62 kDa. The gene shows strong homology to previously cloned delta 9 fatty acid desaturase genes from yeast and rat. Homology includes three histidine boxes characteristic for membrane-bound desaturases and a cytochrome b5 domain responsible for electron transport. The delta 9 desaturase gene has a high G+C content of 61% and displays a codon usage different from that of Saccharomyces cerevisiae, but similar to that of the basidiomycete Schizophyllum commune. Expression of the delta 9 desaturase gene of C. curvatus ATCC 20509 was studied in the presence of different fatty acids in the growth medium. Repression of desaturase mRNA signals was found if fatty acids with a double bond at the delta 9 position were present. Fatty acids with a double bond at another position (delta 10 or delta 6) or saturated fatty acids had no effect on the transcription of the cloned gene.

Amino Acid Sequence↗
PubMed1999

Production of sophorolipids from whey: development of a two-stage process with Cryptococcus curvatus ATCC 20509 and Candida bombicola ATCC 22214 using deproteinized whey concentrates as substrates.

In order to produce sophorolipids from whey, thereby lowering the lactose content and biological oxygen demand, a two-step batch cultivation process was developed including medium sterilization by filtration. In the first step, whey was sterilized by a combination of crossflow and sterile filtration. Because the sophorolipid-producing yeast Candida bombicola ATCC 22214 was not able to use lactose as a carbon source directly, the oleaginous yeast Cryptococcus curvatus ATCC 20509 was grown on deproteinized whey concentrates (DWC). With 1:1 diluted DWC-20, lactose was consumed as the carbon source and biomass (24 g/l dry weight content) as well as single-cell oil (SCO, 10 g/l) were produced. The cultivation broth was disrupted with a glass bead mill and it served as medium for growth (29 g cell dry mass/l) and sophorolipid production (12 g/l) of the yeast C. bombicola.

Acetylation↗
PubMed2000

Uniparental mitochondrial transmission in sexual crosses in Cryptococcus neoformans.

Restriction fragment length polymorphism (RFLP) in the large ribosomal RNA region of the mitochondrial DNA (mtDNA) was developed as a genetic marker for investigating mitochondrial transmission in sexual crosses of the human pathogenic basidiomycetous yeast Cryptococcus neoformans. Strain JEC20 of C. neoformans var. neoformans (mat a) was mated with six strains of C. neoformans var. grubii (mat alpha). Successful mating was indicated by the formation of hyphae and basidiospores. These basidiospores were examined for mtDNA RFLP genotypes. All 570 basidiospores examined from the six crosses showed the mtDNA genotype of strain JEC20. The failure to recover the C. neoformans var. grubii mtDNA in any cross indicates that the C. neoformans var. grubii mtDNA is either selectively eliminated in the newly formed dikaryon or selectively excluded in the immediate dikaryotic hyphae of the newly formed dikaryon.

Crosses, Genetic↗
PubMed2002

Mitochondrial DNA polymorphisms in the human pathogenic fungus Cryptococcus neoformans.

This study examined mitochondrial DNA (mtDNA) restriction site polymorphisms among 416 strains of the human pathogenic yeast Cryptococcus neoformans from the United States and Japan. The strains included 378 serotype A, 14 serotype D, 18 serotype AD, two serotype B, and two strains whose serotype could not be determined using current commercial monoclonal antibodies. Portions of two genes were examined: (1) the mitochondrial large ribosomal RNA gene (mtLrRNA) and (2) the NADH dehydrogenase subunit 2 ( ND2). To screen for polymorphisms among the 416 strains, the endonuclease MaeIII was used to digest the PCR-amplified mtLrRNA gene fragment and three endonucleases ( BanI, AluI, MseI) were used to digest the PCR-amplified ND2 gene fragment. Four mtDNA haplotypes were identified among these strains. All strains of serotype A had mtDNA haplotype I, strains of serotype D had haplotype II, and strains of serotype B had haplotypes III and IV. Of the two non-typable strains, one was haplotype I while the other was haplotype II. Among the strains of serotype AD, 14 were haplotype I and the other four were haplotype II. These results were discussed in the context of recent findings regarding the origins of serotype AD strains and the observed uniparental mtDNA inheritance in laboratory crosses between strains of serotypes A and D.

Cryptococcus neoformans↗
PubMed2004

Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals.

Cryptococcus neoformans serotype A strains commonly infect immunocompromised patients to cause fungal meningitis. To understand the basis of serotype A cryptococcal infections in apparently immunocompetent patients, we tested two hypotheses: the strains were naturally occurring hypervirulent pkr1 (PKA regulatory subunit) mutants, or the strains were hybrids with C. neoformans var. gattii strains that normally infect immunocompetent individuals. Analysis of clinical isolates obtained from apparently immunocompetent individuals from three continents revealed that none were pkr1 mutants, but several exhibited phenotypes consistent with perturbations in cAMP signaling. Additionally, none of the strains were unusual hybrids with gattii strains. Except for one strain that was an AD hybrid, all others were serotype A (var. grubii) isolates. Taken together, our findings indicate that the ability of these clinical isolates to infect apparently normal individuals may be attributable to mutations other than pkr1 and/or underlying immune system impairment in patients.

Blotting, Southern↗
PubMed2004

Cryptococcus neoformans mitochondrial genomes from serotype A and D strains do not influence virulence.

Cryptococcus neoformans is an encapsulated pathogenic yeast producing meningoencephalitis. Two primary strains in genetic studies, serotype A H99 and serotype D JEC21, possess dramatic differences in virulence. Since it has been shown that mitochondrial gene expression is prominent at the site of the infection and there are significant differences between mitochondrial gene structure and regulation between the serotype A and D strains, this study used AD hybrids to move serotype A and D mitochondria under different genomic influences. When the serotype D MATa strain is involved in the mating crosses, there is uniparental transmission of mitochondrial DNA, but with the serotype A MATa strain, mitochondrial DNA can be inherited from either parent and recombination in the mitochondrial genome may also occur. In virulence studies between serotype A and D strains, it was found that the primary genetic control of the virulence composite for growth in the central nervous system is encoded in the nuclear DNA and not through mitochondrial DNA.

Animals↗
PubMed2004

A Sch9 protein kinase homologue controlling virulence independently of the cAMP pathway in Cryptococcus neoformans.

The polysaccharide capsule is one of the established virulence factors in Cryptococcus neoformans that provides a barrier against the host-mediated immune response. Mutation of the gene encoding the Saccharomyces cerevisiae Sch9 protein kinase homologue resulted in cells with enlarged capsules in C. neoformans. Capsule production was abrogated in sch9 pka1 double mutants, indicating that protein kinase A (PKA) signaling is still necessary for capsule formation in sch9 mutants. The sch9 mutant also exhibited increased thermal tolerance, a phenotype similar to sch9 mutant strains of S. cerevisiae. In addition, the sch9 mutant was attenuated in mating and the highly encapsulated cells were attenuated in virulence, in contrast to the pkr1 mutant, lacking the regulatory subunit of protein kinase A, that produced similarly enlarged capsules yet was increased in virulence. Interestingly, the virulence for the sch9 mutant strain could be restored by introduction of a pkr1 mutation; and the sch9 pkr1 mutant strain was dramatically increased in size and capsule thickness, suggesting that Sch9 and PKA function via different targets involved in virulence. Our findings support a model in which Sch9 modulates capsule formation and contributes to the virulence of C. neoformans both independently of and in conjunction with the cAMP-PKA pathway.

Animals↗
PubMed2007

The mating type-specific homeodomain genes SXI1 alpha and SXI2a coordinately control uniparental mitochondrial inheritance in Cryptococcus neoformans.

In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type alpha (MATalpha) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type alpha (MATalpha)-specific gene SXI1a, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATalpha parent, and both the MATa and the MATalpha parents. In addition, progeny from same-sex mating between MATalpha strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1alpha and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.

Cryptococcus neoformans↗
PubMed1997

Variability of karyotypes and RAPD types in genetically related strains of Cryptococcus neoformans.

Variation in karyotypes and RAPD patterns of genetically related strains of Cryptococcus neoformans were analyzed. Capsular and filamentous mutants usually differ in their karyotypes from wild-types, but the RAPD patterns were found to be similar. Karyotype differences were observed in most heterothallic matings, but RAPD patterns remained identical. After self-sporulation of a diploid strain, minor chromosomal length polymorphism and minor changes in the RAPD types occurred. Three mechanisms, either alone or in combination, may in varying degrees contribute to the karyotype variation of C. neoformans: (1) mitotically induced changes; (2) karyotype changes as a result of meiotic recombination, and (3) mutagen-induced changes. The present data do not support the meiotic maintenance hypothesis, which claims that the amount of CLP generated is inversely proportional to the frequency of meiosis.

Cryptococcus neoformans↗
PubMed1998

Construction of stable episomes in Cryptococcus neoformans.

We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene (ADE2/URA5) used as a selection marker.

Blotting, Southern↗
PubMed2005

Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep.

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.

Animals↗
PubMed2006

Phenol degradation by immobilized cold-adapted yeast strains of Cryptococcus terreus and Rhodotorula creatinivora.

Three strains were isolated from hydrocarbon-polluted alpine habitats and were representatives of Cryptococcus terreus (strain PB4) and Rhodotorula creatinivora (strains PB7, PB12). All three strains synthesized and accumulated glycogen (both acid- and alkali-soluble) and trehalose during growth in complex medium containing glucose as carbon source and in minimal salt medium (MSM) with phenol as sole carbon and energy source. C. terreus strain PB4 showed a lower total accumulation level of storage compounds and a lower extracellular polysaccharides (EPS) production than the two R. creatinivora strains, PB7 and PB12. Biofilm formation and phenol degradation by yeast strains attached to solid carriers of zeolite or filter sand were studied at 10 degrees C. Phenol degradation by immobilized yeast strains was always higher on zeolite compared with filter sand under normal osmotic growth conditions. The transfer of cells immobilized on both solid supports to a high osmotic environment decreased phenol degradation activity by all strains. However, both R. creatinivora PB7 and PB12 strains maintained higher ability to degrade phenol compared with C. terreus strain PB4, which almost completely lost its phenol degradation activity. Moreover, R. creatinivora strain PB7 showed the highest ability to form biofilm on both carriers under high osmotic conditions of cultivation.

Adaptation, Physiological↗