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Melanin synthesis has been associated with virulence for the human pathogenic fungus Cryptococcus neoformans. Recent evidence indicates that C. neoformans cells synthesize melanin during infection and that this pigment protects the fungus against immune defense mechanisms.
Cryptococcus neoformans was cultured on one occasion from the oropharyngeal washings in 1 of 820 and from the interdigital areas of the feet in 6 of 723 healthy persons. Among the positive persons were two school children, two laboratory workers, one gardener, one plumber, and one printing press worker. Repeat cultures were negative in all seven positive persons; serological tests for cryptococcal antigen/antibody were negative in three. The results demonstrate that C. neoformans may occur as a transient inhabitant of the oropharynx or skin of healthy people.
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.
The interaction of the opportunistic fungus Cryptococcus neoformans with swine microglia was studied in vitro in the presence and absence of anti-CD14 monoclonal antibodies. In the absence of anti-CD14 antibodies, 36% of microglia had phagocytized nonopsonized, encapsulated cryptococci after 2 hr incubation (effector-to-target ratio, 1:50). Preincubation of microglia with anti-CD14 antibodies resulted in a 63% reduction of phagocytosis. These findings suggest that CD14 receptors facilitate uptake of nonopsonized C, neoformans by resident macrophages within the brain.
D-proline assimilation and CGB tests were performed on 233 Cryptococcus neoformans isolates from Brazil. The results disclosed a high sensitivity, D-proline 99-13% and CGB 99.57%, for screening of the varieties. D-proline assimilation test revealed two false negative results for 5.1% of 39 strains of var. gattii, and no false positive results were observed in 194 strains of var. neoformans. No false results and only one doubtful result were found on the CGB medium.
We investigated the pathogenesis of pulmonary Cryptococcus neoformans infection and passive Ab efficacy in mice deficient in inducible NO synthase (NOS2(-/-)) and the parental strain. Parental mice lived significantly longer than NOS2(-/-) mice after intratracheal infection, despite having a higher lung fungal burden. Administration of Ab reduced lung CFU in both NOS2(-/-) and parental mice, but prolonged survival and increased the inflammatory response only in parental mice. Ab administration was associated with increased serum nitrite and reduced polysaccharide levels in parental mice. Eosinophils were present in greater numbers in the lung of infected NOS2(-/-) mice than parental mice, irrespective of Ab administration. C. neoformans infection in NOS2(-/-) mice resulted in significantly higher levels of IFN-gamma, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha than parental mice. Ab administration had different effects on infected NOS2(-/-) and parental mice with respect to IFN-gamma, monocoyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha levels. Ab administration increased lung levels of IFN-gamma in parental mice and reduced levels in NOS2(-/-) mice. The results indicate that NO is involved in the regulation of cytokine expression in response to cryptococcal pneumonia and is necessary for Ab efficacy against C. neoformans in mice. Our findings indicate a complex relationship between Ab efficacy against C. neoformans and cytokine expression, underscoring the interdependency of cellular and humoral defense mechanisms.
Leukocyte recruitment to the site of infection by the encapsulated yeast Cryptococcus neoformans is critical for clearance of the infection. We review data from our lab that chemokines, such as the CC chemokines MCP-1 and MIP-1alpha, are important mediators of leukocyte recruitment during C. neoformans infection. In addition, studies in CC chemokine receptor knockout mice have demonstrated that CCR2 and CCR5 are required not only for leukocyte recruitment but also for other aspects of immune response development and innate imunity to C. neoformans.
A new medium effectively differentiates B and C serotypes of Cryptococcus neoformans from A and D serotypes on the basis of resistance to low concentrations of cycloheximide and assimilation of glycine as the sole carbon source.
In February and March 1984, Cryptococcus neoformans was detected in the manure and ambient air of a volery in the Berlin Zoo in which a Palm Cockatoo (Probosciger aterrimus) was kept. Both times, 5 colony-forming units of Cr. neoformans could be isolated on the differential medium, Guizotia abyssinica creatinine agar, from 40 1 of air aspirated by the Reuter Centrifugal Sampler (RCS). The absence of a concurrent growth of moulds was found to be of special importance for the optimal isolation of Cr. neoformans on the above mentioned agar for purposes of epidemiological research into airborne dissemination of the fungus. The advantages and disadvantages of 0.1% biphenyl to inhibit concurring growth of moulds are discussed. The control of habitats and foci of Cr. neoformans in zoos and similar establishments is considered a necessity, to prevent inhalatory exposure of susceptible, e.g. immuno-compromised persons.
The capsule of Cryptococcus neoformans can undergo dramatic enlargement, a phenomenon associated with virulence. A prior study that used Ab to the capsule as a marker for older capsular material concluded that capsule growth involved the intermixing of new and old capsular material with displacement of older capsular polysaccharide towards the surface. Here we have revisited that question using complement (C), which binds to capsular polysaccharide covalently, and cannot redistribute by dissociation and binding at different sites. The experimental approach involved binding of C to cells with small capsules, inducing capsule growth, and following the location of C relative to the cell wall as the capsule enlarged. C remained close to the cell wall during capsule growth, indicating that capsule enlargement occurred by addition of new polysaccharide near the capsule edge. This conclusion was confirmed by an independent method that employed radioactive metabolic labelling of newly synthesized capsule with 3H-mannose followed by gradual capsular stripping with gamma-radiation. Capsule growth proceeded to a certain size, which was a function of cell size, and was not degraded when the cells were transferred to a non-inducing medium. During budding, an opening appeared in the capsule of the mother cell that permitted the nascent bud to separate. Scanning EM suggested that a physical separation formed between the capsules of the mother and daughter cells during budding, which may avoid mixture between both capsules. Our results indicate that C. neoformans capsular enlargement also occurs by apical growth and that budding results in capsular rearrangements.
A method is described for the serotyping of Cryptococcus neoformans based on direct analysis of culture supernatants for the major type-specific capsular antigen, glucuronoxylomannan. Factor sera prepared by absorption of polyclonal rabbit antisera (Iatron Laboratories, Inc., Tokyo, Japan) or selected anti-C. neoformans monoclonal antibodies were used in a dot enzyme assay to detect the presence of antigen.
Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis as the most frequent clinical presentation in immunocompromised patients, mainly in people infected by HIV. This fungus is an environmental encapsulated yeast, commonly found in soil enriched with avian droppings and plant material. A total of 290 samples of pigeon and the other avian droppings, soil, ornamental trees and vegetable material associated with Eucalyptus trees were collected to study environmental sources of Cryptococcus species in Goiânia, Goiás State. The determination of varieties, serotypes and the susceptibility in vitro to fluconazole, itraconazole and amphotericin B of C. neoformans isolates were performed. C. neoformans var. grubii (serotype A) was found in 20.3% (36/177) of pigeon dropping samples and in 14.3% (5/35) of samples of Eucalyptus. None of the environmental isolates of C. neoformans showed in vitro resistance to three antifungal agents. The knowledge of major route for human cryptococcal infection (inhalation of infectious particles from saprophytic sources) and a total of 60 C. neoformans isolates obtained from AIDS patients with cryptococcal meningitis between October 2001 and April 2002 justify the study of the habitats of these yeasts as probable sources of cryptococcosis in this city.
Cryptococcus neoformans grown with L-dopa was melanized and was less susceptible to amphotericin B. The results suggest that in vivo and in vitro susceptibilities to amphotericin B may differ.
Serial isolates of Cryptococcus neoformans from patients with chronic infection can exhibit minor karyotype changes as a result of chromosome length polymorphism (CLP). This study investigated whether serial C. neoformans isolates with CLP from 4 patients with AIDS exhibited biologic and phenotypic differences. CLP permits the identification of serial isolates in murine mixed infection. The parameters studied were virulence in mice, capsule size, colony morphology, melanization, protease production, MICs of antifungal drugs, and growth rates in vitro. Two parameters of virulence in mice were studied: persistence in tissue and survival time after lethal infection. Serial C. neoformans isolates were shown to differ in ability to persist in vivo, virulence in a murine infection model, in vitro growth rates at 37 degreesC, and capsule size. Melanin and protease production and MICs of antifungal drugs were comparable for serial isolates. These observations suggest microevolution of C. neoformans during human infection. This process may allow the fungal population to change, escape eradication by the immune system, and thus cause chronic infections.
The antifungal susceptibilities of 1,811 clinical isolates of Cryptococcus neoformans obtained from 100 laboratories in 5 geographic regions worldwide between 1990 and 2004 were determined. The MICs of amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, and ravuconazole were determined by the National Committee for Clinical Laboratory Standards broth microdilution method. Isolates were submitted to a central reference laboratory (University of Iowa) from study centers in Africa (5 centers, 395 isolates), Europe (14 centers, 102 isolates), Latin America (14 centers, 82 isolates), the Pacific region (7 centers, 50 isolates), and North America (60 centers, 1,182 isolates). Resistance to amphotericin B, flucytosine, and fluconazole was < or = 1% overall. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) ranged from 35% in North America to 68% in Latin America. Similarly, only 75% of isolates from North America were susceptible to fluconazole (MIC, < or = 8 microg/ml) compared to 94 to 100% in the other regions. Isolates remained highly susceptible to amphotericin B (99% susceptibility at a MIC of < or = 1 microg/ml) over the entire 15-year period. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) increased from 34% in 1990 to 1994 to 66% in 2000 to 2004. Susceptibility to fluconazole (MIC, < or = 8 microg/ml) increased from 72% in 1990 to 1994 to 96% in 2000 to 2004. Voriconazole, posaconazole, and ravuconazole all were very active (99% of isolates susceptible at MIC of < or = 1 microg/ml) against this geographically diverse collection of isolates. We conclude that in vitro resistance to antifungal agents used in the treatment of cryptococcosis remains uncommon among isolates of C. neoformans from five broad geographic regions and has not increased over a 15-year period.
A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.
A 115-kDa exoantigen produced by Cryptococcus neoformans recognized by the previously described murine monoclonal antibody 7C9 has been purified from culture filtrate by a combination of membrane ultrafiltration, isoelectric focusing, and preparative gel electrophoresis. It is produced in late-log-phase cultures and is present in greater amounts in cultures grown at 25 degrees C than in those grown at 37 degrees C. Recognition of the antigen by 7C9 on immunoblots is abolished by the proteolytic enzymes papain and trypsin. The antigen is a glycoprotein bearing N-linked oligosaccharides, of which mannose is an important constituent. It does not appear to have proteolytic activity and is acidic, with a pI of 3 to 3.2. Its relationship to previously described C. neoformans mannoprotein is unclear since 7C9 shows only very weak cross-reactivity with a purified sample of the latter. Sera from patients infected with C. neoformans exhibited strong recognition of the glycoprotein as shown by immunoenzyme development of Western immunoblots, indicating its possible significance as a marker of disease.
Life-threatening infections caused by the encapsulated fungal pathogen Cryptococcus neoformans have been increasing steadily over the past 10 years because of the onset of AIDS and the expanded use of immunosuppressive drugs. Intricate host-organism interactions make the full understanding of pathogenicity and virulence of C. neoformans difficult. We discuss the current knowledge of the characteristics C. neoformans must possess to enter the host and establish progressive disease: basic growth requirements and virulence factors, such as the polysaccharide capsule; shed products of the organism; melanin production; mannitol secretion; superoxide dismutase; proteases; and phospholipases.