[Cryptococcus neoformans, var. neoformans, Cryptococcus neoformans var. gattii, causative agents of cryptococcosis].
Explore the source record for details and available documents.
Acquired Immunodeficiency Syndrome↗
SEARCH · Search PubMed
Results for “Cryptococcus neoformans”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Light microscopy of basidia, basidiospores, and nuclei in spores and hyphae of Filobasidiella neoformans (Cryptococcus neoformans).
Three hypha-forming strains of Cryptococcus neoformans were induced to form basidia and basidiospores. Light microscopy showed that basidia formed at the ends of terminal hyphal cells and were able to produce from a few to many basidiospores. The morphology of the sexual structures indicated that these strains belonged to the recently described perfect state of C. neoformans, Filobasidiella neoformans. The average dimensions of the basidiospores were 1.9 mum in width by 2.7 mum in length. Giemsa staining revealed that dikaryotic cells were formed in all three strains. Only one strain had both terminal and subterminal dikaryons, indicating functional clamp connections, whereas the two remaining strains had dikaryons restricted to the terminal cells. Basidiospores of two strains were mononucleate, and yeast cell clones derived from single basidiospores of these two strains were able to complete the sexual life cycle, thus indicating their primary homothallic nature.
Cell Nucleus↗
Ultrastructure of septal complex in Filobasidiella neoformans (Cryptococcus neoformans).
Electron microscopy of Filobasidiella neoformans, the perfect state of Cryptococcus neoformans, revealed basidiomycete doliporesepta between hyphal cells and also between clamp connections and adjacent cells. The pore-occluding material was a heterogeneous flattened plate with dark margins and a lighter center, as seen in the species of Filobasidium. Representative basidiomycete parenthesomes were lacking, and endoplasmic reticulum was seen in the dolipore region.
Cell Wall↗
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↗
HIV-1 envelope protein (gp120) inhibits the activity of human bronchoalveolar macrophages against Cryptococcus neoformans.
Cryptococcus neoformans infections are a major cause of morbidity and mortality for HIV-infected persons. Containment of the initial respiratory inoculation to the lung appears defective in patients with AIDS despite the low burden of HIV in bronchoalveolar macrophages. We have studied the fungistatic activity of human bronchoalveolar macrophages (BAM) cultured with an encapsulated strain of C. neoformans in the presence of pooled human serum. We observed 51.6% fungistasis after 24 h of culture. Fungistasis was diminished if the pooled human serum was heat-inactivated but was not affected by anticryptococcal capsular IgG. HIV envelope protein (gp120) has been shown to interfere with lymphocyte activation in vitro. We studied the effects of gp120 on BAM function and found that fungistatic activity was inhibited 25% (p < 0.001). Although binding of yeasts was not affected, gp120 inhibited the internalization of bound yeasts by 46% (p = 0.025). These experiments indicate that gp120 decreases the internalization and fungistasis of C. neoformans by human BAM, and they suggest a mechanism to explain how a small number of HIV-1-infected cells in the lung could impair the containment of C. neoformans.
Bronchoalveolar Lavage Fluid↗
Six-hour pigmentation test for the identification of Cryptococcus neoformans.
Cryptococcus neoformans colonies can be identified within 6 h using paper disks containing caffeic acid and ferric citrate. Indentification is based on the development of a dark brown pigment. Saprophytic Cryptococcus species and common clinically isolated yeasts do not develop the brown color. The concentration of the reagents and the method of storage of the impregnated paper disks are critical for the rapid and specific development of the pigment.
Caffeic Acids↗
Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.
Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.
Cryptococcus↗
Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans.
Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14-28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10(6) cryptococci, while no mortality was observed in mice exposed to 10(3) or 10(4) cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.
Animals↗
Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans.
Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significant threat to immunocompromised individuals, particularly those with HIV/AIDS. Effective genetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectively selected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affect cell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirming its applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.IMPORTANCECryptococcus neoformans is a type of fungus that can cause serious illnesses in people who have weakened immune systems, like those with HIV/AIDS. To better study this fungus and find new treatments, scientists need tools to change its genes in precise ways. However, the current tools available for this are somewhat limited. This research introduces a new tool called the phosphite dehydrogenase gene/phosphite system, which does not rely on antibiotics to work. It uses a gene from a different bacterium that helps select and grow only the fungus cells that have successfully incorporated new genetic information. This is particularly useful because it does not interfere with the normal growth of the fungus or the features that make it harmful (like its ability to change shape or produce protective coatings). By making it easier and more effective to manipulate the genetics of C. neoformans, this tool opens up new possibilities for understanding how this fungus operates and for developing therapies to combat its infections. This is crucial for improving the treatment of infections in vulnerable populations.
Cryptococcus neoformans↗
Molecular and genetic analysis of URA5 transformants of Cryptococcus neoformans.
Cryptococcus neoformans var. neoformans ura5 mutants were transformed with linearized or circular plasmids containing the C. neoformans orotidine monophosphate pyrophosphorylase gene. Following electroporation, randomly isolated transformants were analyzed for the mitotic and meiotic stability of uracil prototrophy. All stable transformants tested showed nonspecific ectopic integration. Uracil prototrophy in these transformants was stable through meiosis. Some of the stable transformants showed integration of both URA5 and vector sequences, while others lacked any vector sequences. Unstable transformants exhibited the presence of an autonomously replicating plasmid which had undergone significant sequence rearrangement. The autonomously replicating plasmid in the transformants was observed to be the same size or smaller than the transforming plasmid, was maintained in a linear form, and had acquired a genomic sequence(s) with homology to a sequence(s) on all the chromosomes. The conservation of a 300-bp sequence at the 5' end of the URA5 gene was observed in all the rearranged plasmids. These results suggest mechanisms of plasmid maintenance in C. neoformans that are different from those reported for other yeasts. The ura5 mutant was significantly less virulent than the wild type. The transformants did not recover virulence regardless of prototrophic stability.
Chromosome Mapping↗
Catecholamines and virulence of Cryptococcus neoformans.
Cryptococcus neoformans was unable to utilize catecholamines (epinephrine, norepinephrine, or dopamine) as sole carbon or nitrogen sources. Therefore, catecholamines are not essential growth factors for this fungus and the brain is not a preferred nutritional niche for its growth with regard to catecholamines. To establish whether the brain is a survival niche for C. neoformans and to explain the role of phenoloxidase as a virulence factor, a wild-type strain that had phenoloxidase activity and mutants which lacked it were exposed to an epinephrine oxidative system, and the survival of both strains was tested. The oxidative system contained epinephrine as an electron donor, Fe3+ as the catalytic transition metal ion, and hydrogen peroxide as an electron acceptor. The wild-type strain was found to be resistant to this oxidative system, whereas under the same conditions the mutant strain was susceptible and its survival decreased at a rate of 4 logs per h. Damage to high-molecular-weight DNA seems to be a causative factor of cell death after exposure of the mutants to the oxidative system. These results suggest that C. neoformans may survive in the brain because of its ability to utilize catecholamines for melanogenesis and thus neutralize the harmful effects of catecholamines which are manifested in the presence of hydrogen peroxide and transition metal ions. The role of phenoloxidase in resistance to the epinephrine oxidative system is also discussed.
Catechol Oxidase↗
Cloning of 18S and 25S rDNAs from the pathogenic fungus Cryptococcus neoformans.
Cryptococcus neoformans is an important pathogenic fungus that has been classified as a basidiomycete. Little is known of the molecular genetics of this fungal pathogen. To begin such studies, we devised a procedure for extraction of DNA from cryptococci; this method involved the use of the cell wall-active enzyme NovoZym 234. Using cloned rDNA of Saccharomyces cerevisiae as a probe, we identified homologous restriction fragments in a Southern blot of digested C. neoformans DNA. An 8.6-kilobase HindIII fragment that hybridized with the yeast rDNA probe was ligated with the vector pBR322 and cloned into Escherichia coli. When the fragment was used as a probe, it hybridized to the 18S and 25S rRNAs of C. neoformans in Northern (RNA) blots of native and denatured RNA. It bound at high stringency only weakly to the rRNAs of the ascomycete S. cerevisiae. The locations of the genes for 5/5.8S, 18S, and 25S subunits in the cloned fragment were identified with labeled rRNA of these different types.
Blotting, Northern↗
Ecology, life cycle, and infectious propagule of Cryptococcus neoformans.
Cryptococcus neoformans is a biotrophic smut-like fungus, and the epidemiology of cryptococcosis can mainly be explained by exposure to an infective aerosolised inoculum. For C neoformans var gattii it is postulated that the principal infectious propagule is the basidiospore and that exposure to Eucalyptus camaldulensis, the host tree, is required to initiate infection in man and animals. C neoformans var gattii may have been exported from Australia by infected seeds of E camaldulensis containing dormant dikaryotic mycelium of the fungus. For C neoformans var neoformans both the basidiospore and desiccated encapsulated yeast cells are postulated to act as infectious propagules, the basidiospores showing a seasonal distribution in association with an as yet unidentified host plant, and the encapsulated yeast cells dispersed from accumulations of dried bird (mainly pigeon) droppings which act as a year-round vector.
Algorithms↗
Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans.
Cryptococcus neoformans is an encapsulated yeast-form fungus which causes pulmonary and meningeal infections preferentially in the immunocompromised host. It is thought that cell-mediated immunity is important for acquired resistance against cryptococcosis with activated macrophages as the final effector cells. However, specific polysaccharides in the capsule of C. neoformans protect the fungus from adherence to phagocytes and from subsequent phagocytosis. We have studied extracellular killing of C. neoformans by IFN-gamma-activated macrophages and their products. Murine bone marrow-derived macrophages stimulated with rIFN-gamma for 24 h were able to effectively suppress the growth of C. neoformans and the effect of IFN-gamma was augmented by LPS. Killing of C. neoformans was also achieved by cell-free supernatants from bone marrow-derived macrophages stimulated with IFN-gamma plus LPS. Our results indicate that killing of C. neoformans by activated macrophages is independent from toxic oxygen radicals and mediated by secreted protein(s) of apparent molecular mass of 15 and 30 kDa. These findings indicate that activated macrophages play a major role in host defense, although the fungus resists phagocytosis and remains in the extracellular milieu.
Animals↗
Production of diagnostic pigment by phenoloxidase activity of cryptococcus neoformans.
Cryptococcus neoformans produces brown pigmented colonies when grown on agar media made from an extract of potatoes and carrots, broad beans (Vicia faba), or Guizotia abyssinica seeds. Since other yeasts do not produce the pigment, these media are useful as differential isolation media for C. neoformans. Similar specific pigment was produced by C. neoformans on chemically defined agar media which contained six different substrates of phenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.10.3.1) an enzyme which catalyses the oxidation of o-diphenols to melanin. Substrates were incorporated singly into the media and included L-3, 4-dihydroxyphenylalanine (L-DOPA), chlorogenic acid, protocatechuic acid, catechol, norepinephrine, and 3-hydroxytyramine hydrochloride (dopamine). No pigment was produced on media without substrate. Phenoloxidase activity in (NH(4))(2)SO(4) precipitates of C. neoformans cell-free extract was assayed by measuring increases in absorbance at 480 nm produced in solutions of L-DOPA. This reaction showed oxygen uptake and was effectively inhibited by copper chelators, but not by catalase. The enzyme also oxidized the five other substrates which induced pigment formation. Electron micrographs of cells incubated in L-DOPA showed deposition of the pigment in the cell wall.
Agar↗
Facilitated isolation, purification, and analysis of glucuronoxylomannan of Cryptococcus neoformans.
Cryptococcus neoformans was cultured in a chemically defined medium. The culture was adjusted to 0.25% formaldehyde or autoclaved after 5 days of growth at 35 degrees C, and a cell-free supernatant was obtained by centrifugation. Solid calcium acetate was added to the supernatant to give a 5% solution, and the pH was adjusted to approximately 5 with glacial acetic acid. The polysaccharide (PS) was precipitated by the addition of 3 volumes of 95% ethanol. The PS was dissolved in 0.2 M NaCl, and insoluble calcium salts were solubilized by the addition of several drops of glacial acetic acid. The PS solution was treated by ultrasonic irradiation for 15 min. This concurrently decreased the molecular weight of the PS and reduced the viscosity of the solution. The ultrasonically irradiated PS was precipitated by differential complexation with hexadecyltrimethylammonium bromide at 23 degrees C, the complex was dissolved in 1 M NaCl, and the glucuronoxylomannan was precipitated by adding 3 volumes of ethanol. The glucuronoxylomannan was dissolved in 1 M NaCl and then ultrasonically irradiated for 2 h to reduce the molecular mass to a limiting value of approximately 100 kDa (GXMS). The purified GXMS was centrifuged, dialyzed, and finally recovered by lyophilization. GXMS was chromatographed on DEAE-cellulose at reasonable concentrations without the complication of high solution viscosity. The sugar composition and structure of GXMS were determined by gas-liquid chromatography, permethylation gas-liquid chromatography-mass spectrometry, and 13C nuclear magnetic resonance spectroscopy. The improved solution characteristics of GXMS were ideal for the determination of its chemical and serological properties.
Cryptococcus neoformans↗
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↗
Pigment production by Cryptococcus neoformans and other Cryptococcus species from aminophenols and diaminobenzenes.
Cryptococcus neoformans and other Cryptococcus species can produce pigment(s) from many aminophenol and diaminobenzene compounds. Pigment production from these compounds is similar to the conversion of diphenols to melanin by C. neoformans. Several pigmentation patterns (resulting in the identification or grouping of Cryptococcus species) have been observed by using diaminobenzene and aminophenol compounds as substrates. The most common pigmentation pattern observed was pigment production by both C. neoformans and C. terreus. In contrast to the diphenols, only two aminophenols (4-hydroxymetanilamide and 3-aminotyrosine) were found to be highly specific as substrates. They allowed only C. neoformans to produce pigment. When 4-aminosalicylic acid was the substrate, a unique pattern was observed because only C. terreus, C. diffluens, and C. albidus produced pigment. Finally, a pattern was observed in which C. neoformans produced large amounts of pigment from aminophenol and diaminobenzene compounds, whereas the other Cryptococcus species produced smaller amounts. A simplified scheme with three substrates resulted in the identification of C. terreus and C. neoformans as well as two groups of other Cryptococcus species, group I (C. albidus and C. diffluens) and group II (C. laurentii and C. luteolus).
Aminobenzoates↗