RESUMO
The cell cycle pattern of the pathogenic basidiomycetous yeast Cryptococcus neoformans differs from that of the ascomycetous budding yeast Saccharomyces cerevisiae. To clarify the cell cycle control mechanisms at the molecular level, homologues of cell cycle control genes in C. neoformans were cloned and analyzed. Here, we report on the cloning and characterization of genes coding for CDK1 cyclin homologues, in particular, the C. neoformans G1 cyclin. We have identified three putative CDK1 cyclin homologues and two putative CDK5 (PHO85) cyclin homologues from the genome. Complementation tests in an S. cerevisiae G1 cyclin triple mutant confirmed that C. neoformans CLN1 is able to complement S. cerevisiae G1 cyclin deficiency, demonstrating that it is a G1 cyclin homologue. Interestingly, cells deleted of the single Cdk1-G1 cyclin were viable, demonstrating that this gene is not essential. However, it exhibited aberrant budding and cell division and a clear delay in the initiation of DNA synthesis as well as an extensive delay in budding. The fact that the mutant managed to traverse the G1 to M phase may be due to the activities of Pho85-related G1 cyclins. Also, that C. neoformans had only a single Cdk1-G1 cyclin highlighted the importance of keeping in order the commitment to the initiation of DNA synthesis first and then that of budding, as discussed.
Assuntos
Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/fisiologia , DNA Fúngico/biossíntese , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Cryptococcus neoformans/citologia , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Teste de Complementação Genética , Genoma Fúngico , Viabilidade Microbiana , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Cryptococcus neoformans was studied in detail by freeze-substitution and serial ultrathin sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar shaped. The dumbbell-shaped SPBs were 228-365 nm long with amorphous spheres on each end, each sphere being 78-157 nm in diameter. The bar-shaped SPBs were 103-260 nm long and 32-113 nm thick. They consisted of filamentous materials. The dumbbell-shaped SPBs were more frequent (61%) than the bar-shaped SPBs. The bar-shaped SPBs may be regarded as dumbbell-shaped SPBs whose spherical parts became sufficiently small. There seemed to be no relationship between the SPB shape and the cell cycle stage of G1-G2, since both types of SPB appeared not only in unbudded cells but also in budded cells and their appearance seems to be random. It is not clear at present whether morphological changes between dumbbell- and bar shapes have any physiological function. The SPB tended to be localized away from the nucleolus (141 degrees +/- 44 degrees), but localized randomly to the bud (97 degrees +/- 50 degrees). The present study highlights the necessity of observing a large number of micrographs in three dimensions to describe accurately the ultrastructure of the SPB in yeast.
Assuntos
Nucléolo Celular/ultraestrutura , Cryptococcus neoformans/ultraestrutura , Interfase , Fuso Acromático/ultraestrutura , Nucléolo Celular/metabolismo , Tamanho Celular , Cryptococcus neoformans/metabolismo , Substituição ao Congelamento , Humanos , Microscopia Eletrônica , Fuso Acromático/metabolismoRESUMO
Cryptococcus neoformans is an opportunistic human pathogen belonging to basidiomycetous fungi and has unique properties in cell cycle progression. In the present study, dynamics of the spindle pole body (SPB) during the cell cycle was examined using freeze-substitution and serial thin-sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar-shaped in G1 through G2 phases. At the beginning of prophase, globular elements of the SPB enlarged, associated with numerous cytoplasmic microtubules, and separated on the nuclear envelope. At prometaphase, the SPBs entered the nuclear region by breaking a part of the nuclear membrane, were located at the isthmus, and were associated with numerous nuclear microtubules. The nuclear division process was carried out in the daughter cell, though the nucleolus remained in the mother cell. At anaphase, one half of the nucleus returned to the mother cell. At telophase, the SPB element was extruded back to the cytoplasm from the nuclear region. By analyzing serial sections of 63 cells, duplication of the SPB was found to take place in the early G1 phase. Thus, the location, structure, and duplication cycle of the C. neoformans SPB are different from those of Saccharomyces cerevisiae, but have similarities to those of Schizosaccharomyces pombe.
Assuntos
Cryptococcus neoformans/ultraestrutura , Organelas/ultraestrutura , Animais , Proteínas de Ciclo Celular , Microscopia Crioeletrônica/métodos , Substituição ao Congelamento/métodos , HumanosRESUMO
Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0-3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards beta-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.
Assuntos
Ácidos/farmacologia , Antifúngicos/farmacologia , Autólise , Parede Celular/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/fisiologia , Parede Celular/ultraestrutura , Quitina/metabolismo , Microscopia Crioeletrônica , Cryptococcus neoformans/ultraestrutura , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Microscopia de Contraste de Fase , Polissacarídeos/metabolismo , beta-Glucanas/metabolismoRESUMO
We have identified a number of ecto-glycanases (glycosylhydrolases) associated with the capsule and/or the cell wall of Cryptococcus neoformans. The enzyme activities detected included alpha-mannosidase, alpha-, and beta -glucosidase, alpha-, and beta-galactosidase, beta-xylosidase, beta-glucuronidase, and endo-beta-1,3-glucanase. Small portions of the enzymes were also secreted into the growth medium. Cell-wall associated endo-beta-1,3-glucanases exhibited highest activity in the acidic range between pH 2.5 and 5.0. The products of laminarin hydrolysis by the enzymes located on the cell surface were glucose and beta-1,3-linked glucooligosaccharides. The same products were released from isolated cell walls incubated in the buffer. Endo-beta-1,3-glucanase activity extracted from the cell surfaces by mild sonication consisted of six isoforms separable by isoelectric focusing. In spite of the presence of the whole array of glycanase activities on the cell surfaces, capsular polysaccharides released from C. neoformans cells into the growth medium were practically metabolically stable. From the defined polysaccharides tested, only laminarin (beta-1,3-glucan) and to some extent also mixed-linkage beta-1,3/beta-1,4-glucan and/or 4-O-methyl-D-glucurono-D-xylan were able to support the yeast growth. The activities of majority of identified ecto-glycanases were low when the yeast was grown on glucose but were considerably elevated when the cells were grown on glycerol indicating that their synthesis is regulated by catabolite repression.
Assuntos
Parede Celular/metabolismo , Cryptococcus neoformans/metabolismo , Glicosídeo Hidrolases/metabolismo , Parede Celular/enzimologia , Cryptococcus neoformans/enzimologia , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , IsoenzimasRESUMO
In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. The timing of budding in C. neoformans was shifted to later cell cycle points with progression of the growth phase of the culture. Similarly, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G2 phase and cause accumulation of cells after DNA synthesis, but before commitment to budding. The C. neoformans homologue of the main cell cycle control gene CDC28/Cdc2 was isolated using degenerate RT-PCR. The full-length coding region was then amplified using primers to target the regions around the start and stop codons. The gene was called CnCdk1 and was found to have high homologies to S. cerevisiae CDC28 and S. pombe cdc2. To determine its function, its ability to rescue S. cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2-CnCdk1 construct exhibited growth at the restrictive temperature. Results of the sequence analysis and the ability of CnCdk1 to complement the S. cerevisiae cdc28-ts mutations support its assumed role as the CDC28/cdc2 homologue in C. neoformans.
Assuntos
Quinases relacionadas a CDC2 e CDC28/genética , Cryptococcus neoformans/genética , Ciclo Celular , Cryptococcus neoformans/citologia , DNA Fúngico/biossínteseRESUMO
The opportunistic fungal pathogens Candida inconspicua and C. norvegensis are very rarely isolated from patients and are resistant to fluconazole. We collected 38 strains of the two microorganisms isolated from Europe and Japan, and compared the polymorphism of the rRNA intergenic spacer (IGS) and internal transcribed spacer (ITS) regions, antifungal drug susceptibility, and extracellular enzyme production as a potential virulence factor. While the IGS sequences of C. norvegensis were not very divergent (more than 96.7% sequence similarity among the strains), those of C. inconspicua showed remarkable diversity, and were divided into four genotypes with three subtypes. In the ITS region, no variation was found in either species. Since the sequence similarity of the two species is approximately 70% at the ITS region, they are closely related phylogenetically. Fluconazole resistance was reconfirmed for the two microorganisms but they were susceptible to micafungin and amphotericin B. No strain of either species secreted aspartyl proteinase or phospholipase B. These results provide basal information for accurate identification, which is of benefit to global molecular epidemiological studies and facilitates our understanding of the medical mycological characteristics of C. inconspicua and C. norvegensis.
Assuntos
Antifúngicos/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Candida/efeitos dos fármacos , DNA Espaçador Ribossômico/análise , Farmacorresistência Fúngica , Fluconazol/farmacologia , Lisofosfolipase/metabolismo , Anfotericina B/farmacologia , Sequência de Bases , Candida/classificação , Candida/enzimologia , Candida/genética , Equinocandinas , Variação Genética , Lipopeptídeos , Lipoproteínas/farmacologia , Micafungina , Dados de Sequência Molecular , Peptídeos Cíclicos/farmacologia , RNA Ribossômico/genética , Virulência/genéticaRESUMO
A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR. A subsequent search in the C. neoformans genome database identified several sequences similar to CDC28/cdc2. A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR. The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1. To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium. Results of the sequence analysis and the fact that CnCdk1 is able to complement the S. cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C. neoformans.
Assuntos
Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/isolamento & purificação , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Sequência de Aminoácidos , Sequência de Bases , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , Genes Fúngicos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Filogenia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Cryptococcus neoformans was grown first to OD 4 under moderate aeration, then diluted 2.5 times with fresh medium, and grown under limited aeration for 5 h. Oxygen concentration decreased from 5-6 mg l(-1) to 1.5 mg l(-1) 1 h after the shift to limited aeration, and remained at a similar level thereafter. In all the eleven strains examined the shift caused unbudded G(2)-arrest in more than half of the cells. In three strains more than 80% of the cells were arrested in unbudded G(2), and, therefore they were selected for synchrony experiments. After being shifted to extensive aeration again, the cells resumed growth by synchronous budding, followed by synchronous nuclear division. This method has turned out to be a good tool to prepare synchronized culture in C. neoformans, especially when a large amount of synchronized cells is needed. This is worthy of attention, since synchronous cultures after release from G(2)-arrest have not been reported yet in any yeast species.
Assuntos
Ciclo Celular/fisiologia , Cryptococcus neoformans/citologia , Aerobiose , Divisão Celular/fisiologia , Cryptococcus neoformans/crescimento & desenvolvimento , Fase G2/fisiologia , Microscopia de Contraste de Fase , Consumo de OxigênioRESUMO
Cryptococcus neoformans is an opportunistic human pathogen, which infects the central nervous system causing the fatal disease, meningitis. In order to understand the genetic background of this human pathogen, the basic molecular manipulation techniques of deletion, overexpression, and so on have been developed. URA5, a gene encoding orotate phosphoribosyltransferase, has frequently been used to introduce foreign gene fragments by complementing ura5 mutant strains, which are not, however, stable; reversion to uracil prototroph is thus frequently observed on selective condition. The high possibility of reversion makes it inconvenient to use this mutation to identify appropriate transformants and thus, manipulation in molecular genetics. We report here the isolation of a stable ura5 mutant of C. neoformans, designated as TAD1, by eliminating the URA5 gene by homologous recombination using the biolistic DNA delivery system. The availability of the stable ura5 mutant offers the advantage that no spontaneous reversion occurs so that a satisfactory rate of homologous recombination can be achieved. The strain will allow efficient genomic analysis in C. neoformans.
Assuntos
Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidade , Deleção de Genes , Genes Fúngicos/genética , Orotato Fosforribosiltransferase/genética , DNA Fúngico , Genoma Fúngico , Instabilidade Genômica , Meningite Fúngica/microbiologia , Recombinação GenéticaRESUMO
Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.
Assuntos
Amônia/química , Fermentação , Fusarium/metabolismo , Acetato-CoA Ligase/química , Trifosfato de Adenosina/química , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Etanol/metabolismo , Etanol/farmacologia , Cinética , Lisina/química , Microscopia Eletrônica , Modelos Químicos , Mutação , Nitratos/química , Nitratos/farmacologia , Nitritos/química , Oxigênio/metabolismo , Plasmídeos/metabolismo , Fatores de TempoRESUMO
A strain of Histoplasma capsulatum var. duboisii (deposited as IFM 50954 in Chiba University) was isolated from the cerebrospinal fluid of a female Ugandan patient infected with HIV. The isolate had in vitro urease activity on Christensen's urea agar slants, although the common belief is that H. capsulatum var. duboisii is urease negative, and is, considered one of the characteristic markers that distinguishes the three varieties of H. capsulatum. Forty H. capsulatum var. capsulatum, five H. capsulatum var. duboisii, and five H. capsulatum var. farciminosum isolates were evaluated for urease activity on Christensen's urea agar slants and for other qualitative and quantitative urease assays of activity. All 50 isolates of H. capsulatum used in this study were positive for urease activity, suggesting that urease activity may be universal characteristic of H. capsulatum. We also compared the urease activity and pathogenicity of seven H. capsulatum isolates that convert into yeast-form cells. Although isolate IFM 50954 showed moderate virulence in mice and moderate urease activity among tested H. capsulatum isolates, there was no correlation between level of urease activity and pathogenicity. In addition, scanning electron microscopy revealed that some microconidia of isolate IFM 50954 formed "double-cell" configurations that were attached to each other by narrow bases.
Assuntos
Infecções por HIV/microbiologia , Histoplasma/isolamento & purificação , Animais , Feminino , Histoplasma/patogenicidade , Humanos , Japão , Masculino , Camundongos , Camundongos Endogâmicos , Uganda/etnologia , Urease/análiseRESUMO
The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Exophiala dermatitidis was studied in detail. The SPB was located on the outer nuclear envelope and was 342 +/- 86 nm long in a haploid strain. It consisted of two disk elements that measured 151 +/- 43 nm in diameter and 103 +/- 17 nm in thickness, connected by a rod-shaped midpiece that measured 56 +/- 20 nm in length and 37 +/- 9 nm in diameter. There were considerable variations in size and morphology of interphase SPB. Some disk elements appeared spherical but others were more flattened, and there was variation in electron density. A few SPBs did not have the midpiece. The SPB of a diploid strain was 486 +/- 118 nm long, thus significantly bigger than that of the haploid strain. The SPB tended to be localized away from the nucleolus (110 +/- 48 degrees), but close to the bud (78 +/- 45 degrees). The present study highlights the necessity of observing a large number of micrographs in three-dimensions to describe accurately the ultrastructure of the SPB in yeast.
Assuntos
Nucléolo Celular/metabolismo , Exophiala/citologia , Interfase , Fuso Acromático/metabolismo , Animais , Nucléolo Celular/ultraestrutura , Tamanho Celular , Exophiala/genética , Exophiala/fisiologia , Humanos , Fuso Acromático/ultraestruturaRESUMO
The morphologies, numbers, sizes and volumes of all organelles and cell components identified on ultrathin sections of aerobically grown exponential phase yeast cells of Exophiala dermatitidis in G1 phase were examined by freeze-substitution fixation and serial ultrathin sectioning. The cell wall consisted of three layers and occupied approximately 22% of the cell volume. The nucleus was approximately 1.8 microm in diameter and occupied approximately 7% of the cell volume. There was only one nucleolus in the nucleus and it occupied approximately 16% of the nuclear volume. There were 17-52 mitochondria per cell, occupying 7-12% of the cell volume. Five to ten endoplasmic reticula were present per cell; these occupied only 0.2% of the cell volume and did not form a network. There were 1-4 vacuoles per cell and they occupied 4-10% of the cell volume. Storage material was round and electron transparent and occupied 4-11% of the cell volume. The cytosol occupied 43-53% of the cell volume. The Golgi apparatus, spindle pole body, autophagosomes, multivesicular bodies, lipid bodies, microtubules and microfilaments occupied approximately 1% of the cell volume in total. About 200,000 ribosome particles, 1000 glycogen granules and several tens of microtubules (average length 0.78 microm) were present per yeast cell. The membranes of this yeast could be classified into three groups by their appearance and thickness. This is the first report, to our knowledge, that analysed all the components in the yeast cell quantitatively and in three dimensions, and provides fundamental information for understanding various aspects of cell biology.
Assuntos
Exophiala/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Núcleo Celular/ultraestrutura , Parede Celular/ultraestrutura , Exophiala/citologia , Substituição ao Congelamento , Humanos , Microscopia Eletrônica/métodos , Microtomia/métodos , Organelas/ultraestrutura , Tamanho da PartículaRESUMO
Organization of the cytoskeleton was studied in the ascomycetous black yeast Aureobasidium pullulans, an opportunistic human pathogen, in an effort to present it as a potential target of antifungal therapy. Long cytoplasmic microtubules, extending along the hyphae from the base to the growing apex, were the dominant structures in multinucleate interphase cells. Before mitosis these microtubules disappeared and were replaced by intranuclear spindles. This reorganization of microtubules occurred along the whole length of hypha before synchronous division of the nuclei. Actin cytokinetic rings were rarely seen. Cortical actin in the form of patches accumulated in areas of cell wall growth, i.e. in the hyphal apex and near the occasionally formed septum. Actin cables were not seen. During synchronous conidiogenesis, the cytoplasmic microtubules extended along developing conidia, and actin patches lined their subcortical areas. Actin rings were formed regularly at the base of uninuclear conidia. Microtubule inhibitor methyl benzimidazol-2-ylcarbamate disintegrated the microtubules, and inhibited nuclear division, development of hyphae and conidiogenesis. Actin inhibitor Cytochalasin D induced swelling of hyphal apexes and developing conidia. This inhibitory activity ceased after 5 to 12 h when the occasional septa appeared and conidiogenesis was completed. The lack of unicellular organization in multinucleate hyphae of A. pullulans seems be related to a rarity of F-actin structures: i.e. absence of actin cables, the lack of actin cytokinetic rings in particular, resulting in the uncoupling of the nuclear division from cytokinesis; the association of both processes is, however, retained during conidiogenesis.
Assuntos
Actinas/metabolismo , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/ultraestrutura , Microtúbulos/ultraestrutura , Infecções Oportunistas/microbiologia , Ascomicetos/patogenicidade , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Microscopia Eletrônica , Micoses/microbiologiaRESUMO
The structure of a budding cell of the pathogenic yeast Exophiala dermatitidis was observed in three dimensions after freeze-substitution, serial ultrathin sectioning and computer reconstruction. The nucleus occupied about 10% of the cell volume. The spindle pole body was composed of two disk elements connected by an intervening midpiece, and occupied about 0.01% of the cell volume. The cell wall consisted of an inner transparent layer, a middle electron-opaque layer, and an outer fibrous layer. The mitochondria occupied about 10% of the cell volume. There were numerous mitochondria in the mother cell and the bud, but no 'giant mitochondrion' was seen. The ratio of mitochondrial volume within the bud to the mitochondrial volume of the cell was close to the ratio of bud:cell cytoplasmic volume. The results emphasize the importance of good cryofixation for 'perfect' preservation of yeast cell structure.
Assuntos
Exophiala/ultraestrutura , Substituição ao Congelamento/métodos , Processamento de Imagem Assistida por Computador/métodos , Microtomia/métodos , Exophiala/patogenicidade , Humanos , Microscopia Eletrônica/métodosRESUMO
Serotype, mating type and ploidy of 84 strains of Cryptococcus neoformans isolated from 61 AIDS and 23 non-AIDS patients admitted in a tertiary teaching hospital in Sõo Paulo, Brazil were examined. Among 61 strains isolated from AIDS patients, 60 strains were var. grubii (serotype A). Only one strain was var. gattii (serotype B). No var. neoformans (serotype D) was found. Among 23 strains isolated from non-AIDS patients, 15 were var. grubii (serotype A) and the remaining 8 were var. gattii, all of which were serotype B. Seventy-three of the 75 serotype A strains were the heterothallic alpha type (MATalpha) and the remaining 2 were untypable (asexual). Most of the MATalpha strains (69/73) were haploid and the remaining 4 strains were diploid. Similarly, both of the 2 asexual strains among the 75 serotype A strains were haploid. There were no alpha-mating type (MATalpha) strains among the 84 isolates. All of the 8 var. gattii strains were serotype B and haploid. Among a total of 84 strains tested, neither serotype AD nor serotype D were found. Neither triploid nor tetraploid were found. These results suggest that the serological, sexual and ploidy characteristics in C. neoformans strains isolated from AIDS patients in Sõo Paulo were rather simple, whereas strains isolated from non-AIDS patients presented serotype A and B with predominance of serotype A
Assuntos
Humanos , Infecções Oportunistas Relacionadas com a AIDS , Criptococose , Cryptococcus neoformans , Genes Fúngicos , Ploidias , Brasil , Cryptococcus neoformans , Variação Genética , SorotipagemRESUMO
The spindle pole body of the pathogenic yeast Exophiala dermatitidis was observed during the cell cycle using freeze-substitution and serial ultrathin sectioning electron microscopy. The spindle pole body was located on the outer membrane of the nuclear envelope and consisted of two disk elements connected by an intervening midpiece in G1 through G2 phases. Each disk element was composed of filamentous materials and measured 150 nm in diameter and 100 nm in thickness. The midpiece had higher electron density and measured 60 nm in length and 40 nm in thickness. At the beginning of prophase, each disk element of the spindle pole body enlarged to more than double in size. They were separated on the nuclear envelope, and associated with numerous cytoplasmic microtubules. At mitosis, the spindle pole body entered the nuclear envelope, associated with numerous nuclear microtubules, and was located at the spindle poles. At the end of telophase, it was extruded back into the cytoplasm from the nuclear envelope. Three-dimensional analysis of cells in different cell cycles suggested that duplication of the spindle pole body took place in early G1 phase. Thus, the location, structure, and duplication cycle of the E. dermatitidis spindle pole body were different from those of Saccharomyces cerevisiae.
Assuntos
Exophiala/ultraestrutura , Centro Organizador dos Microtúbulos/ultraestrutura , Ascomicetos/ultraestrutura , Basidiomycota/ultraestrutura , Ciclo Celular , Exophiala/genética , Exophiala/patogenicidade , Substituição ao Congelamento , Fase G1 , Imageamento Tridimensional , Interfase , Cinética , Mitose , Modelos Anatômicos , Filogenia , Prófase , Saccharomyces cerevisiae/ultraestrutura , Schizosaccharomyces/ultraestruturaRESUMO
We have developed a method for preparation of synchronous culture in Cryptococcus neoformans. The method is based on age fractionation of exponentially growing asynchronous culture through differential sedimentation in 10-20% (w/v) lactose gradient. C. neoformans capsule thickness should be reduced to a minimum to ensure most accurate age fractionation, which is necessary to obtain a higher degree of synchrony. The C. neoformans synchronous culture system has revealed important characteristics with respect to cellular morphology, DNA content and cell volume distribution. The method can be used for further cell cycle studies.
Assuntos
Técnicas de Cultura de Células/métodos , Cryptococcus neoformans/crescimento & desenvolvimento , Micologia/métodos , Ciclo Celular , Microscopia de FluorescênciaRESUMO
A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.