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PubMed1976

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↗
PubMed1976

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↗
PubMed2025

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↗
PubMed1976

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↗
PubMed2024

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↗
PubMed1979

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

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

Cryptococcus↗
PubMed1978

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↗
PubMed2025

Unisexual reproduction in the global human fungal pathogen Cryptococcus neoformans.

The human fungal pathogen Cryptococcus species complex (encompassing Cryptococcus neoformans, Cryptococcus deneoformans, and the Cryptococcus gattii species complexes) exhibits diversity in sexual reproduction, including α-a mating, pseudosexual reproduction, as well as unisexual reproduction initiated from a single isolate or between isolates of the same mating type. A central conundrum is that while most Cryptococcus natural populations exhibit significant α mating-type bias, genetic and genomic analyses show recombination occurs in nature. The discovery of unisexual reproduction in C. deneoformans provided insight; however, thus far, unisexual reproduction has never been directly observed in the predominant global pathogenic species C. neoformans. Here, we provide evidence that mutating the RIC8 gene, which encodes a conserved guanine nucleotide exchange factor (GEF) involved in both chaperoning and activating Gα proteins, enables unisexual reproduction in C. neoformans. Additionally, we show that genetic variation in the natural population promotes unisexual reproduction, and unisexual reproduction in C. neoformans involves canonical meiotic recombination. Finally, we found that deletion of both GPA2 and GPA3 in the MATα background leads to self-filamentation without sporulation, suggesting that differential modulation of the Gα proteins, likely involving Ric8, could underlie the switch between different modes of sexual reproduction in Cryptococcus. Our study further highlights that the highly conserved Ric8 GEF can act as an important regulator of cellular development in response to environmental stimuli and could modulate sexual reproduction in nature. We hypothesize that unisexual reproduction occurs much more frequently in nature than currently appreciated, and possibly in other fungi and microbial eukaryotes as well.

Cryptococcus neoformans↗
PubMed1978

Study of the role of pigeons in the dissemination of Cryptococcus neoformans in nature.

Cryptococcus neoformans was recovered from droppings collected within the first 24 h from pigeons experimentally fed with a dose of 5 X 10(6) cells. The fungus proved to multiply well though differently in the sterilized pigeon and chicken excreta seeded with the organism. In both unsterile types of droppings no viable cells of C. neoformans were detected after 4 weeks incubation. Isolated bacterial flora from the intestinal contents of apparently healthy pigeons showed a complete inhibitory effect on the growth of C. neoforms in vitro. It has been concluded that pigeons do not favor multiplication of the fungus in their gut and consequently they do not seem to play an active biological role in dissemination of C. neoformans in nature.

Animals↗
PubMed2025

Meiosis-specific genes play roles in ploidy reduction in Cryptococcus neoformans titan cells.

Cryptococcus neoformans is a fungal pathogen of humans that causes life-threatening meningoencephalitis. During infection, enlarged, polyploid titan cells are produced that promote survival, evade immune cells, and generate diverse progeny. These titan cells divide to produce haploid, aneuploid or diploid daughter cells with enhanced stress tolerance; however, how ploidy reduction occurs is poorly understood. Here, we show that titan cells developed from diploid strains predominantly produce diploid daughter cells with haploid daughters observed infrequently. We further demonstrate that meiosis-specific genes, including DMC1 and SPO11, are critical for stable inheritance of a diploid genome in the daughter cells. Specifically, deletion of these genes in a heterozygous diploid background results in: 1) titan cells with a significantly reduced capacity to produce daughter cells; 2) increased phenotypic variation among daughter cells produced by the titan cells, including traits that could be relevant to cell growth and viability; and 3) daughter cells produced by the titan cells exhibiting high levels of loss of heterozygosity (LOH) and aneuploidy, suggested elevated genome instability. Taken together, these findings demonstrate the importance of meiosis-specific genes in the ploidy reduction process of titan cells derived from a heterozygous diploid background in an important human fungal pathogen.

Dmc1↗
PubMed1979

Response of congenitally athymic (nude) and phenotypically normal mice to Cryptococcus neoformans infection.

A Cryptococcus neoformans infection in congenitally athymic (nude) mice and phenotypically normal heterozygote BALB/c mice was used to determine how T lymphocyte-deficient mice compared with normal mice in restricting proliferation of C. neoformans and to determine whether a correlation exists between delayed-type hypersensitivity and resistance to C. neoformans. Although nude mice displayed the ability to maintain cryptococcal population levels lower than did the phenotypically normal animals during the first 14 days of infection, the resistance was not sufficient to control the infection during the remainder of the 35-day experimental period. Heterozygote mice began to demonstrate positive delayed-type hypersensitivity responses by day 14 postinfection; however, nude mice were unable to mount delayed-type hypersensitivity responses. The appearance of the delayed-type hypersensitivity response in the heterozygote mice was concomitant with the reduced rate of proliferation of C. neoformans observed in those animals from days 14 to 35. Because anticryptococcal antibody titers and cryptococcal antigen levels were equivalent in both groups of mice, T-lymphocyte function was considered to be responsible for the resistance observed in the heterozygote mice. The mechanism by which cryptococcal populations were reduced was not addressed; however, the mouse model system used in these studies would be an ideal tool for studying those mechanisms. Nude mice were able to produce antibodies against cryptococcal cells, indicating that at least one component of C. neoformans is a T-independent antigen. The antibody response was predominantly immunoglobulin M in nude and heterozygote mice. Cryptococcal antigen levels were extremely high in both groups of animals and appeared to increase as C. neoformans cell numbers increased.

Animals↗
PubMed1976

Caprine mastitis due to Cryptococcus neoformans.

The isolation of Cryptococcus neoformans from milk and its demonstration by direct microscopy has been reported in a case of caprine mastitis. This is believed to be the first instance of caprine mastitis in which C. neoformans has been implicated as the etiologic agent.

Animals↗
PubMed1975

Cryptococcus neoformans of saprophytic origin.

Cryptococcus neoformans was isolated on 276 occasions as follows: from pigeon faeces, 159 (45%) of 349 samples; pigeon crops, 39 (50%) of 80 crops; from drinking water, 37 (74%) of 50 samples; from the atmosphere, 3 strains in a contaminated loft. It was also recovered from 38 (19%) of 200 samples of canary faeces. It is possible that C. neoformans survives in the crop of pigeons and occasionally passes to the faeces where it multiplies. The factors influencing its presence are: the irregular cleaning of lofts, overcrowding and air-pollution from poor ventilation.

Air Microbiology↗
PubMed1977

The in vivo incorporation of [32P]-labeled orthophosphate into pyrophosphatidic acid and other phospholipids of Cryptococcus neoformans through cell growth.

Cryptococcus neoformans was cultured in a liquid medium containing [32P]-orthophosphate and harvested at various stages of cell growth. An aliquot of the [32P]-labeled cells was transferred to a nonradioactive medium, and the culture was continued again for some hours. The [32P]-radioactivity composition and the phosphorus composition of individual phospholipids relative to the total phospholipid through the incubation periods were estimated. Although levels of major phospholipids remained constant throughout the cell growth, the distribution pattern of the [32P]-radioactivity of individual phospholipids changed remarkably along with the progress of cell growth. The changing patterns of the specific radioactivities of individual phospholipids through the growth phase demonstrated that phosphatidic acid was one of the most active metabolites in phospholipids and that pyrophosphatidic acid was also metabolically active.

Cryptococcus↗
PubMed1975

Relationship between polyene resistance and sterol compositions in Cryptococcus neoformans.

Six mutants of Cryptococcus neoformans resistant to nystatin and pimaricin and three mutants resistant to amphotericin B were isolated by ultraviolet irradiation techniques from two wild-type strains. The major sterols of the wild-type strains were Delta(7)-ergosten-3beta-ol and ergosterol. All six mutants resistant to nystatin and pimaricin showed either loss of ergosterol and concurrent production of Delta(7, 22)-ergostadien-3beta-ol and Delta(7)-ergosten-3beta-ol, or loss of both the wild-type sterols, with production of Delta(8(9))-ergosten-3beta-ol and Delta(5, 8(9), 22)-ergostatrien-3beta-ol. The mutants producing Delta(7, 22)-ergostadien-3beta-ol and Delta(7)-ergosten-3beta-ol showed relatively low levels of resistance to nystatin and pimaricin, whereas the mutants producing Delta(8(9))-ergosten-3beta-ol and Delta(5, 8(0), 22)-ergostatrien-3beta-ol showed a high level of resistance to either drug. Although highly resistant to amphotericin B, however, the three mutants produced sterol compositions identical to those of the wild types, indicating that the strains acquired resistance other than by alteration of the membrane sterols. The mutants producing Delta(8(9)) and Delta(5, 8(9), 22) sterols were not virulent for mice, showed reduced growth rates at 25 C, and failed to grow at 37 C. The other mutants showed a slightly reduced rate of growth both at 25 and 37 C, and the virulence in mice was slightly reduced in comparison with that of the wild types. These comparisons were on gross observations and were not statistically analyzed.

Animals↗
PubMed1977

Cryptococcosis, with emphasis on the significance of isolation of Cryptococcus neoformans from the respiratory tract.

Cryptococcus neoformans was isolated from 65 patients: 11 had meningitis, 11 had disseminated cryptococcosis without meningitis, and 43 had C neoformans isolated from the respiratory tract. Predisposing conditions and the diagnostic value of cultures from various sites and of the latex agglutination test on cerebrospinal fluid and serum are analyzed for patients with extrapulmonary disease. Nine patients had pleural effusions; the effusion was cultured in six and yielded C neoformans in four. None of 11 deaths among 43 patients with respiratory tract isolates could be attributed to cryptococosis. The 32 survivors were nor treated with antifungal agents. Twenty-six of 43 patients with respiratory isolates had various bronchopulmonary disorders, with chronic obstructive pulmonary diseases and asthma the most common (28 percent). Seven of 28 patients (25 percent) with roentgenographically detected lung lesions had carcinoma of the lung. Roentgenographic evidence of a lung lesion and C neoformans grown from the respiratory tract warrant a further search for carcinoma of the lung.

Adult↗