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Pigment formation for differentiating Cryptococcus neoformans from Candida albicans.

When 2,3- or 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxycinnamic acid are added to growth media, they are converted to a characteristic brown pigment by Cryptococcus neoformans. This pigment formation has hitherto been encountered only when this microorganism was cultivated on media containing Guizotia abyssinica seed. This phenomenon can be used for differentiating Cryptococcus neoformans from Candida albicans. Possible precursors of these o-diphenols (quinic acid, aromatic monohydroxy acids, or tyrosine) do not give rise to the brown pigmentation.

Agar

Virulence, serotype, and molecular characteristics of environmental strains of Cryptococcus neoformans var. gattii.

Four strains of Cryptococcus neoformans var. gattii originating from Eucalyptus camaldulensis, three from Australia and one from San Francisco, were tested for their serotype, virulence for mice, and a number of genetic and molecular characteristics. All were found to be serotype B and showed significantly higher virulence for mice than did the type strains of C. neoformans var. gattii and Filobasidiella neoformans var. bacillispora, which were obtained from human cryptococcosis cases. Electrophoretic karyotypes of the strains from Australia were identical, although they were collected from sites at least 15 to 500 km apart. The electrophoretic karyotype of the strain from San Francisco was the same as that of the Australian isolates except for the mobility of one chromosome. On the contrary, no two isolates of serotype B (of a total of 11) from clinical sources were the same, regardless of their geographic origin. Furthermore, none of the clinical isolates showed a chromosomal banding pattern identical to that of Eucalyptus-originated strains. The Eucalyptus-originated strains failed to form dikaryons when crossed with the tester strains of the two varieties of F. neoformans. Hybridization analysis with a nucleic acid probe (AccuProbe C. neoformans Culture Confirmation Test; Gen-Probe Inc., San Diego, Calif.), however, showed signals of equal intensity for clinical strains and the Eucalyptus-originated strains. Various fungi phylogenetically related to C. neoformans, including a phenol oxidase-positive strain of Cryptococcus laurentii obtained from E. camaldulensis, were negative in the nucleic acid hybridization test. These observations confirm that, in spite of karyotypic differences and the lack of dikaryon formation with the tester strains of F. neoformans, Eucalyptus-originated C. neoformans var. gattii is the same organism as those isolated from cases of human infection. Furthermore, the C. neoformans culture confirmation test using a commercial nucleic acid probe is specific for C. neoformans.

Animals

Rhodotorulic acid from species of Leucosporidium, Rhodosporidium, Rhodotorula, Sporidiobolus, and Sporobolomyces, and a new alanine-containing ferrichrome from Cryptococcus melibiosum.

An examination of 142 strains within 19 genera of yeasts and yeastlike organisms for formation of hydroxamic acids in low-iron culture showed production of hydroxamates by two unclassified strains and by 52 strains among the genera Aessosporon (3 of 3 strains), Cryptococcus (1 of 43), Leucosporidium (3 of 11), Rhodosporidium (4 of 4), Rhodotorula (27 of 39), Sporidiobolus (2 of 2), and Sporobolomyces (12 of 13). Crystalline rhodotorulic acid was isolated in amounts sufficient to account for most or all of the measured hydroxamate in culture supernatants of 16 strains representative of the five last-mentioned hydroxamate-producing genera. A new alanine-containing ferrichrome was isolated from one strain of Cryptococcus melibiosum. Rhodotorulic acid was a major metabolic product of many of the positive strains when grown in low-iron media, and iron was shown to repress its synthesis and excretion into the culture medium. The taxonomic significance of production of hydroxamic acids is described in connection with the position of these yeast species in the subclass Heterobasidiomycetidae.

Alanine

Production of extracellular ribonuclease by yeasts and yeastlike fungi, and its repression by orthophosphate in species of Cryptococcus and Tremella.

A strain of Cryptococcus laurentii and a haploid isolate of Tremella foliacea were shown to produce orthophosphate-repressible ribonuclease in liquid culture. Addition of as little as 1 mM K2HPO4, pH 7.0, completely repressed enzyme production by both fungi. The orthophosphate-repressible enzyme was not produced by other species of the two genera tested. These results, together with other findings, suggest a close phylogenetic relationship between Cryptococcus laurentii and Tremella foliacea. The ability of other yeasts and yeastlike fungi to hydrolyze ribonucleic acid in a solid test medium was assessed. Based on the limited number of organisms available for study, extracellular ribonuclease activity was found in species having close affinity to the Basidiomycetes and in yeasts classified in the ascomycetous genera, Endomycopsis, Hansenula, and Kluyveromyces. Other ascomycetous yeasts did not exhibit extracellular ribonuclease.

Ascomycota

Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species.

Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.

Base Sequence

Epidemiological and immunological studies of Cryptococcus neoformans.

Walter, Jinks E. (University of Pittsburgh, Pittsburgh, Pa.), and Robert W. Atchison. Epidemiological and immunological studies of Cryptococcus neoformans. J. Bacteriol. 92:82-87. 1966.-The complement-fixation fluorescent-antibody test provided a means of differentiating between antibodies of Cryptococcus neoformans and Candida albicans. The test was applied to the sera of 134 pigeon fanciers for detection of antibodies to C. neoformans only. About 22% were positive as compared with 3% of a control group composed of 36 non-pigeon breeders. Positive reactions were observed only with C. neoformans types A and B cells. It was concluded that the pigeon fanciers had presumably been infected previously with C. neoformans type A or type B. Moreover, 48 of 49 isolates of C. neoformans cultured from the pigeon habitats of 72 fanciers studied were serotype A. These findings would seem to substantiate the hypothesis that pigeon habitats serve as reservoirs for human infections, and also that subclinical cryptococcosis is more prevalent than is realized.

Animals

Nucleotide composition of deoxyribonucleic acid of some species of Cryptococcus, Rhodotorula, and Sporobolomyces.

The buoyant density of deoxyribonucleic acid (DNA) from nine species and two varieties of Cryptococcus, three species and two varieties of Rhodotorula, and six species of Sporobolomyces was determined by CsCl density gradient equilibrium centrifugation. Several species were represented by two to four different strains. Expressed in moles per cent of guanine plus cytosine (GC content) the ranges were 49 to 65%, 52 to 70%, and 51 to 65% for Cryptococcus, Rhodotorula, and Sporobolomyces, respectively. For each genus, the GC content was distributed into two discrete groups with averages ranging from 52 to 54 and 60 to 66, respectively. An analysis of these results suggested that the determination of GC content of DNA had a taxonomic value for these yeast genera.

Centrifugation, Density Gradient

Detection of a Trichosporon beigelii antigen cross-reactive with Cryptococcus neoformans capsular polysaccharide in serum from a patient with disseminated Trichosporon infection.

Latex beads coated with anti-Cryptococcus neoformans antibody were agglutinated by serum from a bone marrow transplant recipient having a disseminated infection caused by Trichosporon beigelii. The cryptococcal latex agglutination titer in the serum of the patient rose to 1:2,560 by the time of his death. Necropsy confirmed the disseminated Trichosporon infection and absence of C. neoformans. Cell wall extracts of the isolate of the patient and two additional strains of T. beigelii agglutinated anti-Cryptococcus-coated latex beads. The antigen in the serum of the patient and in the extracts responsible for the agglutination was not destroyed by proteolytic enzymes or heat. A single antigen reactive with rabbit anti-Trichosporon serum could be identified in the serum of the patient and the cell wall extracts by rocket immunoelectrophoresis and crossed immunoelectrophoresis. Rocket immunoelectrophoresis and indirect fluorescent-antibody staining demonstrated that anti-Trichosporon antibody recognized the capsular polysaccharide of C. neoformans.

Adult

Evaluation of a new method for identification of Cryptococcus neoformans which uses serologic tests aided by selected biological tests.

A new method for identifying Cryptococcus neoformans isolates and their serotypes by the slide agglutination test using five kinds of factor sera, with the aid of nitrate reduction, phenol oxidase, and growth at 37 degrees C tests was evaluated by using 36 reference strains and 75 clinical isolates of C. neoformans. The results showed that the reference strains were identified exactly as they were labeled, and clinical isolates were identified as C. neoformans serotypes A, D, and AD. C. neoformans could be distinguished from other Cryptococcus species that cross-reacted with factor sera by their ability to grow at 37 degrees C. These results indicate that the slide agglutination test combined the use of factor sera for isolates which grow at 37 degrees C is a useful method for identification of C. neoformans and their serotypes and that the nitrate reduction test (negative in 100% of the isolates) and the phenol oxidase test (positive in approximately 95% of the isolates) can be used to confirm that the species is C. neoformans.

Agglutination Tests

Rapid clearance of Candida albicans mannan antigens by liver and spleen in contrast to prolonged circulation of Cryptococcus neoformans antigens.

Clearances of mannan antigen from Candida albicans and glucuronoxylomannan antigen from Cryptococcus neoformans were examined in nonimmune rabbits by using self-prepared latex agglutination tests. Injected intravenously, 20 mg of Candida mannan antigen was cleared from the serum with a half-life of approximately 2 h. In contrast, 20 micrograms of Cryptococcus glucuronoxylomannan antigen had a half-life in serum of approximately 24 h. At the latest, 9 h after injection, both antigens were no longer detectable without pretreatment of serum samples with protease and heating to 100 degrees C, thus indicating rapid binding by serum proteins other than immunoglobulins. Candida mannan antigen clearance was also examined in nonimmune mice after intravenous injection of (i) 200 micrograms of Candida mannan antigen, which accumulated in the liver and spleen and persisted for 97 days; (ii) 2 x 10(7) ethanol-killed Candida blastospores, which was accompanied by rapid clearance of mannan from the blood but accumulation of mannan in the liver and spleen and slow clearance from these organs; (iii) 6 x 10(6) viable C. albicans cells (lethal infection), which resulted in a rapid decrease of Candida CFU in the blood, liver, and spleen during the first 8 h, after which blood cultures were negative on day 2 and viable Candida burdens in the liver and spleen persisted at 10(5) CFU/g, whereas Candida mannan antigen continued to circulate in the bloodstream and accumulated in the liver and spleen.

Animals

Distinct STRIPAK subunits drive conserved and subunit-specific signaling programs in Cryptococcus neoformans.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a conserved protein phosphatase 2A (PP2A)-associated signaling hub that integrates kinase-phosphatase networks, yet its roles in human fungal pathogens remain poorly defined. Here, we dissected STRIPAK functions in the opportunistic pathogen Cryptococcus neoformans by combining genetic, genomic, virulence, and phosphoproteomic analyses across mutants lacking individual STRIPAK subunits. Loss of the core STRIPAK components via PPH22, FAR8, FAR9, or FAR11 mutations caused severe defects in growth, stress adaptation, cell cycle progression, and morphogenesis, accompanied by widespread aneuploidy and genome instability. In murine infection models, far11Δ strains were avirulent, whereas far9Δ mutants caused delayed but ultimately fatal disease and underwent host-associated genome remodeling, with recovered isolates exhibiting chromosome 11 amplification despite no consistent in vitro fitness advantage. In contrast, deletion of MOB3 produced a hypervirulent phenotype. mob3Δ cells exhibited enhanced transmigration across an in vitro blood-brain barrier model, increased survival in macrophages, and generated small-cell morphotypes, features associated with increased dissemination. Phosphoproteomic profiling revealed extensive and overlapping phosphorylation changes among core STRIPAK mutants, affecting pathways involved in signaling, cytoskeletal, cell cycle control, chromatin regulation, RNA metabolism, and stress responses. Conversely, mob3Δ mutants displayed a smaller, largely distinct phosphoproteomic signature. Network and functional enrichment analyses highlighted STRIPAK-dependent regulation of TORC2-associated signaling, MAPK/GTPase signaling, autophagy, nuclear transport, RNA processing, DNA replication, and ribosome biogenesis. Together, these findings establish STRIPAK as a coordinator of genome stability, morphological plasticity, stress adaptation, and virulence in C. neoformans, and demonstrate that individual STRIPAK subunits drive shared yet divergent signaling outputs that shape host-pathogen interactions.IMPORTANCEFungal pathogens must rapidly adapt their growth, morphology, and stress responses to survive within the host, requiring precise coordination of cellular signaling pathways. The conserved striatin-interacting phosphatase and kinase (STRIPAK) complex controls key developmental programs in eukaryotes, but its roles in fungal pathogenesis are not fully defined. We previously showed that STRIPAK is important for genome stability, development, and virulence in the opportunistic human fungal pathogen Cryptococcus neoformans. Here, we define how individual STRIPAK subunits differentially regulate fungal morphogenesis, genome plasticity, host adaptation, and virulence, revealing both shared and subunit-specific functions within this conserved signaling complex. Core STRIPAK mutants exhibit severe growth and stress-response defects and attenuation of virulence, whereas loss of the Mob3 subunit promotes hypervirulence by enhancing dissemination and persistence within the host. Phosphoproteomic profiling reveals that individual STRIPAK components exert shared yet distinct control over phosphorylation networks that shape host-pathogen interactions, establishing STRIPAK as a central signaling hub and a potential target for antifungal intervention.

Cryptococcus neoformans

A method for authenticating the fidelity of Cryptococcus neoformans knockout collections.

Gene knockout (KO) strain collections are important tools for discovery in microbiology. Cryptococcus neoformans, a human fungal pathogen, has an available genome-wide gene deletion collection that is widely used by the research community. We uncovered mix-ups in the assembly of the commercially available C. neoformans deletion collection of ~4,700 unique strains acquired by our laboratory. Evidence supporting a mix-up includes RNAseq analysis that identified transcripts for the gene listed as the KO. The mystery was soon solved as this same KO strain lacked RNA transcripts for a different KO strain gene found in the same plate position in an earlier partial KO collection, suggesting a plate swap between two KO collections. Therefore, we developed a quick PCR assay to distinguish the two KO collections based on the size differences between their nourseothricin (NAT)-resistance cassettes, confirmed by genome sequencing. Here, we report that nine of the first 15 plates of the 42-plate our KN99ɑ KO collection had been replaced with the corresponding plates from an earlier partial KO collection. We provide additional evidence that the remaining plates are correct, and the simple authentication method presented here serves as a quick check to identify similar mix-ups in the KO collections.IMPORTANCEGene KO strain collections are important tools for discovery in microbiology. The human fungal pathogen Cryptococcus neoformans has an available genome-wide deletion collection that is widely used by the research community. Here, we report that our KN99ɑ collection is comprised of mixed plates from two independent KO libraries and present a simple authentication method that other investigators can use to distinguish the identities of these KO collections. Above all, this article serves as a reminder to users of the 2015 KO library collection to screen the plates before undertaking large phenotyping experiments.

Cryptococcus neoformans

CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the most common cause of human fungal meningitis and an important model system for studying fundamental eukaryotic biology. Genetic manipulation of this organism relies on three dominant drug resistance markers (nourseothricin acetyltransferase [NAT], neomycin phosphotransferase II [NEO], and hygromycin B phosphotransferase [HYG]) and the recyclable dominant prototrophic marker amdS. With ongoing technological advances that are expanding our ability to explore cryptococcal gene function, contemporary studies often require multiple genetic manipulations in the same strain. Additional dominant selection methods would maximize the utility of these tools by facilitating their combinatorial use. Here, we identify blasticidin S resistance via the blasticidin S deaminase (BSD) or blasticidin S resistance (BSR) markers as a novel dominant selection method for C. neoformans. We further validate phleomycin resistance via the bleomycin resistance gene (BLE) marker as an additional selection method, confirming a study that first established this marker 25 years ago (J. Hua, J. D. Meyer, and J. K. Lodge, Clin Diagn Lab Immunol 7:125-128, 2000, https://doi.org/10.1128/cdli.7.1.125-128.2000). To enable highly efficient CRISPR/Cas9-mediated genome modification, we incorporated these markers, as well as the newly established dominant prototrophic marker ptxD (M. Khongthongdam, T. Phetruen, and S. Chanarat, Microbiol Spectr 13:e01618-24, 2025, https://doi.org/10.1128/spectrum.01618-24), into a vector series that enables the construction of fused marker-sgRNA products via PCR. Altogether, this work expands the number of dominant genetic selection methods for C. neoformans to seven, including five drug selection regimes and two prototrophic methods. The vector series has been deposited at Addgene. IMPORTANCE Cryptococcus neoformans is the top-ranked World Health Organization priority fungal pathogen due to its widespread distribution and inadequate treatment options. Additionally, as a basidiomycete yeast occupying an underexplored branch of the fungal kingdom, this organism is a powerful system for deciphering core eukaryotic biology that is absent in classic model fungi. Defining functions for novel cryptococcal genes is a crucial priority, and the availability of additional genetic selection methods would facilitate these efforts. In this study, we establish blasticidin S resistance as a novel genetic selection method for C. neoformans, and we validate a previous report using phleomycin resistance as such. This work expands the number of reliable dominant selection methods to seven, providing flexibility for the introduction of sequential genetic modifications into single strains.

Cryptococcus neoformans

[The structure of extracellular heteroglycans in various Cryptococcus species].

Extracellular polysaccharides produced by some Cryptococcus species have been structurally investigated. These polymers have identical core structure, which was found to be alpha-1,3-mannan and different degrees of substitution of mannose in the core by xylose and glucuronic acid residues of side chains and different composition of side chains. Heteropolysaccharide from Cr. humicolus, the simplest one, has the same structure as the Cr. neoformans serotype D capsular polysaccharide. The Cr. skinneri polymer proved to be the most branched among Cryptococcus polysaccharides.

Carbohydrate Sequence

[Light and electron microscopic observations of cysts in the brain from an autopsy case of cryptococcus meningitis].

We gave some considerations to the significance of cyst formation in a case of cryptococcus meningitis by examining the cysts themselves and comparing the organisms in the cysts with those disseminated throughout the subarachnoid space by light and electron microscopy. An 18-year-old girl had complained of headache, stiffneck and fever at the onset. These symptoms worsened into confusion without any definite diagnosis, then resulted in an arrest of spontaneous respiration which led to use of respirator for 12 days. The patient died 40 days after the onset. The brain weighed 1440 g and showed moderate swelling with opacity of the leptomeninges, which was very evident over the convexity and around the basal side of the pons. Subarachnoid fresh hemorrhage was also observed around the basal side of the brain stem. Microscopic examination of the subarachnoid space revealed widely disseminated Cryptococcus neoformans varied in size, whose cell wall showed a positive staining reaction to PAS. The organisms had characteristic spicules positively stained with cresyl violet radiating out of the cell body, and were associated with infiltration of lymphocytes, macrophages and polymorphonuclear leukocytes throughout the subarachnoid space. Some portions of arachnoid membrane, dura mater and vessel walls in the subarachnoid space especially the adventitia of the basilar artery were replaced by multiple cysts. The cysts were tightly filled with large numbers of small uniformly sized organisms which often showed budding. These cysts showed no histological evidence for inflammation. Further studies to demonstrate those differences were carried out with electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent

Tracing the evolution and genomic dynamics of mating-type loci in Cryptococcus pathogens and closely related species.

Sexual reproduction in basidiomycete fungi is governed by MAT loci (P/R and HD), which exhibit remarkable evolutionary plasticity, characterized by expansions, rearrangements, and gene losses often associated with mating system transitions. The sister genera Cryptococcus and Kwoniella provide a powerful framework for studying MAT loci evolution owing to their diverse reproductive strategies and distinct architectures, spanning bipolar and tetrapolar systems with either linked or unlinked MAT loci. Building on recent comparative genomic analyses, we generated additional chromosome-level assemblies, uncovering distinct trajectories shaping MAT loci organization. Contrasting with the small-scale expansions and gene acquisitions observed in Kwoniella, our analyses revealed independent expansions of the P/R locus in tetrapolar Cryptococcus, possibly driven by pheromone gene duplications. Notably, these expansions coincided with a pronounced GC-content reduction best explained by reduced GC-biased gene conversion following recombination suppression, rather than relaxed codon usage selection. Diverse modes of MAT locus linkage were also identified, including three previously unrecognized transitions: one resulting in a pseudobipolar arrangement and two leading to bipolarity. All three transitions involved translocations. In the pseudobipolar configuration, the P/R and HD loci remained on the same chromosome but genetically unlinked, whereas the bipolar transitions additionally featured rearrangements that fused the two loci into a nonrecombining region. Mating assays confirmed a sexual cycle in C. decagattii, demonstrating its ability to undergo mating and sporulation. Progeny analysis in K. mangrovensis revealed substantial ploidy variation and aneuploidy, likely stemming from haploid-diploid mating, yet evidence of recombination and loss of heterozygosity indicates that meiotic exchange occurs despite irregular chromosome segregation. Our findings underscore the importance of continued diversity sampling and provide further evidence for convergent evolution of fused MAT loci in basidiomycetes, offering new insights into the genetic and chromosomal changes driving reproductive transitions.

MAT genes

[Pulmonary pseudotumor caused by Cryptococcus neoformans].

Since 1923, eight cases of cryptococcosis have been described in Norway, all with meningeal affection. A distinct, solitary infiltration in the upper lobe of the right lung was discovered in a 68 year-old woman. The lobe was extirpated. Microbiological and histological investigations showed infection with Cryptococcus neoformans. No antimycotic treatment was given. Four months later the patient developed osteomyelitis of the skull due to cryptococcosis, and was treated successfully with amphotericin B and flucytosin. Further investigation of the patient revealed a defect in the T-lymphocyte immune system, but it is uncertain whether this was of any significance for the development of the cryptococcus infection.

Aged

[CSF examination in cryptococcus meningitis].

CSF studies of 14 cases of cryptococcus meningitis revealed: 1. Direct discerning of yeast cells in the blood cell counting chamber, by Indian ink stain, and by cytological examination based on Sayk's technic, all were highly positive in repeated examinations. 2. Morphology of cryptococcus and inflammatory cellular reactions in CSF were investigated, and were quite characteristic. 3. Suppression and destruction of yeast cells were attainable only when doses of amphotericin B were sufficient.

Adult