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PubMed1994

Epidemiology studies of clinical isolates of Cryptococcus neoformans of Japan by restriction fragment length polymorphism.

Previous epidemiological studies of Cryptococcus neoformans infection in Japan showed that only C. neoformans var. neoformans is present and serotype A is the most common with frequencies in excess of 95%. A DNA fingerprinting method, using a genomic DNA probe (UT-4p), has become available recently which discriminates between individual isolates in a population that are morphologically and serologically indistinguishable. Fifty-two serotype A isolates of C. neoformans were obtained from three different institutions (in Nagasaki, Chiba, and Tokyo) in Japan. Only two of these strains were isolated from AIDS patients and one from pigeon excreta. Of the nine reported finger-printing patterns in serotype A, only three types (IV, V and VII) were observed in Japanese isolates. Pattern IV was almost exclusively observed in Nagasaki isolates (21/22) with only one of pattern VII. In Chiba, however, patterns VII and IV appeared to be equally distributed. In Tokyo, patterns IV and V (which included two isolates from AIDS patients) were observed at similar frequencies. Restriction fragment length polymorphism analysis of four isolates of serotype AD showed a typical serotype A pattern which also contained a serotype D-specific band. This finding suggests the independence of serotype AD. These data could enhance the survey of the epidemiology of cryptococcosis.

Cryptococcosis↗
PubMed2005

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans.

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.

Antibodies, Fungal↗
PubMed2005

Cryptococcus neoformans {alpha} strains preferentially disseminate to the central nervous system during coinfection.

Cryptococcus neoformans is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (gattii, grubii, and neoformans). Each variety manifests differences in epidemiology and disease, and var. grubii strains are responsible for the vast majority of human disease. In previous studies, alpha strains were more virulent than congenic a strains in var. neoformans, whereas var. grubii congenic a and alpha strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var. grubii was further characterized in a panel of model systems. Congenic var. grubii a and alpha strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between a and alpha congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections, a and alpha strains are equivalent in peripheral tissues but alpha cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic a and alpha strains in the most common pathogenic variety and suggest an explanation for the prevalence of alpha strains in clinical isolates.

Animals↗
PubMedDate not supplied

Reduced recovery of a Cryptococcus neoformans adherence mutant from a rat model of cryptococcosis.

Stable mutants of Cryptococcus neoformans (strain CSF-1) induced by treatment with ultraviolet light and nitrosoguanidine were isolated that demonstrated reduced adherence to glial cells in culture. Adherence of the mutants, as measured by a radiometric assay, was reduced by 50-70% of that attained for the parent CSF-1 strain. The adherence mutants appeared to be phenotypically similar to the CSF-1 strain. However, all but one mutant (designated as CSF-23) demonstrated slightly slower growth rates than the wild-type strain. The CSF-1 and CSF-23 strains were injected intravenously and intratracheally into normal rats and rats immunosuppressed by cyclophosphamide treatment, and the organ distribution and recovery of viable yeasts determined over 2-96 h. During this relatively short period of observation the majority of the yeasts were localized in the lungs. By either route of injection, the recovery of the CSF-23 adherence mutant was reduced by as much as 90% of that obtained for the wild-type strain. The results indicated that host cell adherence may be important for the persistence of cryptococci in tissue and that further studies with the adherence mutants are warranted.

Animals↗
PubMed2001

First isolation of Cryptococcus neoformans var. gattii from a native jungle tree in the Brazilian Amazon rainforest.

Cryptococcus neoformans var. gattii was isolated for the first time from decaying wood in a hollow of a native jungle tree Guettarda acreana, in a wild area of an Amazon rainforest island, in Brazil. The presence of this variety in a virgin environment without either anthropic action or introduced vegetation is discussed with regard to the common knowledge of Cr. neoformans ecology.

Brazil↗
PubMed1994

Biochemical basis for the killing of Cryptococcus neoformans by rat peritoneal cells.

The biochemical basis of peritoneal cell cytotoxicity for Cryptococcus neoformans was studied by measuring the killing of the yeast by peritoneal resident cells and peritoneal exudate cells obtained from normal and proteose-peptone-injected animals, respectively. Both cell populations killed C. neoformans to an equivalent extent after 3 h incubation. Exudate cells showed anti-cryptococcal activity from the first hour of incubation, while no killing was observed with resident cells before 3 h. Both cell populations triggered a respiratory burst in response to opsonized C. neoformans as indicated by the fact that killing of the yeast was inhibited by scavengers of reactive oxygen intermediates (ROI). C. neoformans susceptibility to H2O2 and hydroxyl radicals in cell-free systems is demonstrated by incubating a yeast suspension with different concentrations of H2O2 and Fenton's reagents, respectively. These results suggest that oxygen metabolites play an active role in C. neoformans killing.

Animals↗
PubMed1984

In vitro human lymphocyte responses to Cryptococcus neoformans. Evidence for primary and secondary responses in normals and infected subjects.

Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. To study the role of immune lymphocytes in protection and recovery from this infection, in vitro lymphocyte proliferative responses to whole killed organisms were characterized in normal controls and in patients with cryptococcosis. Several differences were found between these two groups. All normal individuals were found to have significant proliferative responses to cryptococci in vitro; however, patients recovering from infection had accelerated and augmented responses. Patients' T cells, but not control T cells, were found to produce interleukin 2 (IL 2) during initial stimulation in vitro. After in vitro priming, mononuclear cells from normal controls rechallenged with cryptococci in vitro demonstrated accelerated kinetics and IL 2 production similar to that of cells from recovering patients. Proliferative responses in normals required T cells and antigen-presenting cells, and resulted in an increase in the proportion of T8+ and Tac+ cells in the responding population by the ninth day of primary stimulation. An increase in Tac+ cells, but not T8+ cells, was found by the fourth day of secondary stimulation. These results demonstrate in vivo acquisition of T cell responsiveness to the organism in this small group of recovering patients, and in vitro acquisition of T cell recognition of this antigen by normal T cells during primary stimulation. The data suggest that these responses represent in vitro priming to cryptococci and indicate a role for the T8+ lymphocyte subpopulation, both unusual findings for previously described in vitro responses to soluble antigens.

Antigens, Fungal↗
PubMed1995

Tissue localization of Cryptococcus neoformans glucuronoxylomannan in the presence and absence of specific antibody.

During infection, Cryptococcus neoformans capsular glucuronoxylomannan (GXM) is released into tissues, where it may be associated with a variety of deleterious immunological effects. Relatively little is known about the organ distribution and cellular localization of GXM antigen. Intravenous administration of GXM to rats resulted in persistent serum levels which declined with a half-life of 14.3 h in the first 74 h and 3 h thereafter, coincident with the appearance of serum antibodies to GXM. GXM was sequestered primarily in spleen tissue, with localization to marginal zone and follicular cells. Administration of the murine immunoglobulin G1 monoclonal antibody (MAb) 2H1 resulted in > 99% reduction in serum GXM level within 3 h. MAb 2H1 administration resulted in liver GXM deposition, with cellular localization primarily to Kupffer cells. GXM was also found in the spleens of MAb 2H1-treated rats, with localization to the marginal zones and follicles. Endotracheal administration of GXM resulted in low serum levels, with lung tissue having the highest GXM organ levels, localized primarily to alveolar macrophages. The results indicate that (i) intravenous administration to rats produced persistent serum GXM levels with a half-life similar to that found in mice and rabbits; (ii) endotracheal administration of GXM resulted in low serum levels; (iii) in the absence of specific antibody, GXM organ deposition occurs primarily in the spleen and is localized primarily to marginal zone macrophages; (iv) in the presence of specific immunoglobulin G1 antibody, GXM organ deposition occurs primarily in the liver and is localized primarily to Kupffer cells; and (vi) reticuloendothelial cells sequester GXM in the presence and absence of specific antibody.

Animals↗
PubMed2004

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Acetyltransferases↗
PubMed2003

Differences in outcome of the interaction between Cryptococcus neoformans glucuronoxylomannan and human monocytes and neutrophils.

Disseminated infections by the opportunistic yeast Cryptococcus neoformans are characterized by accumulation in tissues of glucuronoxylomannan (GXM), the major component of the capsular polysaccharide. We investigated binding, uptake, and disposal of GXM by peripheral blood neutrophils and monocytes, and the effect of GXM uptake on phagocytic cell function. GXM was efficiently bound and internalized by both types of phagocytic cells, with maximal loading at 50 microg/ml, a GXM concentration found in serum and cerebrospinal fluid of some cryptococcosis patients. However, substantial differences were noted in the kinetics for uptake by macrophages and neutrophils. Whereas neutrophils rapidly ingested limited amounts of GXM and then expelled or degraded it after 1 h of incubation, macrophages demonstrated continuous intracellular accumulation for up to 1 week of incubation. Accumulation of GXM by neutrophils was accompanied by reduced anticryptococcal activity, suggesting one more mechanism for virulence enhancement by the major capsular component of C. neoformans.

Antigens, Fungal↗
PubMed1996

Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D. French Cryptococcosis Study Group.

Differences between the two varieties of Cryptococcus neoformans with regard to geographic distribution, epidemiology, and even pathogenicity have been described. None between C. neoformans variety neoformans serotypes A and D have been reported. We reviewed 452 cases of cryptococcosis diagnosed in France, where serotype D is responsible for 21% of the infections. Univariate analysis showed that the frequency of serotype D infections was significantly higher among patients > 60 years of age, those born in Europe, those receiving corticosteroid therapy, and those who had skin lesions. In the multivariate analysis, the risk of serotype D infection was significantly higher for patients with skin lesions, those receiving corticosteroid therapy, and those living in certain regions in France. It was significantly lower for patients with meningitis those coming from Africa, and females. Among the 350 human immunodeficiency virus-infected patients, the risk was significantly higher for those > 60 years old, those who were intravenous drug abusers, and those with skin lesions. The risk was significantly lower for patients from Africa and in cases of meningitis. These results suggest that individual and environmental factors can be associated with the serotype of the infecting strain of C. neoformans.

AIDS-Related Opportunistic Infections↗
PubMed1991

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↗
PubMedDate not supplied

[Determination of the minimum inhibitory concentration of fluconazole against Cryptococcus neoformans].

The minimum inhibitory concentration of fluconozole was determined in 36 strains of Cryptococcus neoformans var. neoformans by a casitone broth microdilution method, taking into account that it is one of the most used antifungal agents in our country for the treatment of cryptococcal meningitis, the second opportunistic mycosis in order of frequency and that with the highest lethality among AIDS patients. The minimum inhibitory concentration of the studied strains ranged 0.125 to more than 64 micrograms/mL for a geometrical mean of 2.38 micrograms/mL. 50% of the strains were inhibited with 4 micrograms/mL and the least concentration that inhibited 90% was 16 micrograms/mL. It is stressed the importance of continuing this type of in vitro studies to detect the appearance of resistance of C. neoformans to antifungal agents.

Antifungal Agents↗
PubMed1984

Binding of cryptococcal polysaccharide to Cryptococcus neoformans.

Radioiodinated cryptococcal polysaccharide was used to study binding of the soluble polysaccharide to encapsulated and non-encapsulated cryptoccoci. Binding of polysaccharide to non-encapsulated cryptococci occurred rapidly over a 30-min period and was largely complete after 2 h. Bound, labeled polysaccharide was slowly eluted from Cryptococcus neoformans after the addition of unlabeled polysaccharide, indicating reversibility of binding. Non-encapsulated cryptococci bound polysaccharide in two ways. Specific binding to the yeast was saturable by ca. 82 ng of polysaccharide per 10(6) yeast cells. Nonspecific binding also occurred which was not saturable under the conditions used in our experiments. Phagocytosis of the non-encapsulated yeast strain was inhibited when the specific binding was ca. 50% saturated. Binding of polysaccharide to an encapsulated strain showed nonspecific, nonsaturable binding, but little specific binding occurred. Presumably the specific binding sites were saturated in the encapsulated strain. Polysaccharides obtained from a hypocapsular mutant (A61) and a normally encapsulated strain competed effectively with labeled serotype D polysaccharide for binding sites on non-encapsulated cryptococci and had identical phagocytosis-inhibiting properties. Similarly, polysaccharides from all four cryptococcal serotypes competed effectively with labeled serotype D polysaccharide for binding sites on the non-encapsulated strain, and all four polysaccharides inhibited phagocytosis of non-encapsulated Cryptococcus neoformans. Unmodified, de-O-acetylated, carboxyl-reduced, periodate-oxidized and reduced (polyalcohol), and Smith-degraded polysaccharides competed with labeled polysaccharide for binding sites on the cell. The unmodified, de-O-acetylated and carboxyl-reduced polysaccharides inhibited phagocytosis of non-encapsulated cells, but the polyalcohol and Smith product were unable to inhibit phagocytosis.

Animals↗
PubMed2003

Cryptococcus neoformans virulence is enhanced after growth in the genetically malleable host Dictyostelium discoideum.

Cryptococcus neoformans is an encapsulated, environmental fungus that can cause life-threatening meningitis. Pathogenicity of C. neoformans for macrophages and vertebrate hosts may be a mechanism selected in evolution for protection against environmental predators. In this study, we investigated whether Dictyostelium discoideum could serve as an alternate host for C. neoformans. D. discoideum has a defined genetic system which provides significant advantages for the study of fungus-amoeba interactions. Our results show that D. discoideum is susceptible to infection with C. neoformans and that the interactions are similar to those described previously for this fungus with macrophages and Acanthamoeba castellanii. Acapsular C. neoformans cells did not replicate when coincubated with D. discoideum. However, incubation of acapsular C. neoformans with D. discoideum mutants defective in myosin VII synthesis resulted in infection, validating the concept that avirulent organisms can be virulent in impaired hosts even at the unicellular level. Phagocytosis of C. neoformans by D. discoideum could be inhibited with capsule-specific antibodies and various sugars. Passage of an encapsulated C. neoformans strain through D. discoideum cultures increased virulence and was accompanied by larger capsules and faster time to melanization. These results add to the evidence implicating soil ameboid predators as important factors for the maintenance of C. neoformans virulence in the environment and suggest that D. discoideum promises to be an extremely useful system for studying the interaction of C. neoformans with phagocytic cells.

Animals↗
PubMed1991

Killing of Cryptococcus neoformans strains by human neutrophils and monocytes.

The susceptibility of various strains of Cryptococcus neoformans to killing by human polymorphonuclear leukocytes (PMNs) and monocytes was investigated. Five previously characterized strains of C. neoformans serotype A, a capsule-free mutant, and six recent clinical isolates were compared. PMNs and monocytes were isolated from normal peripheral blood and allowed to adhere to the flat-bottom wells of microtiter plates. Yeast cells of C. neoformans were added in the presence of normal human serum, and the plates were incubated at 37 degrees C. After 4 h, killing was determined by comparing the quantitative plate counts of viable yeast cells in experimental wells with counts in control wells containing yeast cells in the absence of leukocytes. No appreciable growth of yeast cells occurred in the wells during the incubation period. Both PMNs and monocytes effectively killed yeast cells at effector-to-target ratios as low as 1:1, although monocytes failed to kill the capsule-free strain 602 at a 1:1 ratio. With 9 of 12 strains, PMNs killed C. neoformans more effectively than did monocytes. Significant interstrain variation in killing occurred for both monocytes and PMNs, and the recent, clinical isolates were more resistant to killing by monocytes and PMNs than were the previously characterized strains. The extent to which different strains were killed by monocytes and PMNs was not consistently related to the size of the capsule or the entire cell. Normal PMNs and monocytes are remarkably effective in killing strains of C. neoformans in the absence of specific antibody and appear to constitute a significant defense mechanism in the peripheral circulation.

Cryptococcus neoformans↗
PubMed2005

Difficulty in diagnosing chronic meningitis caused by capsule-deficient Cryptococcus neoformans.

We report a case of chronic meningitis due to capsule-deficient Cryptococcus neoformans which could not be diagnosed by routine morphological and immunological cerebrospinal fluid (CSF) examination. Repeated CSF examination and culture did not disclose the fungal body, and the cryptococcal antigen tests were always negative. Culture of ventricular fluid showed non-encapsulated cultured cells which were stained positively by indirect immunofluorescence using diluted sera from another patient diagnosed with cryptococcal meningitis. Inoculation of dispersed colonies into mice resulted in encapsulated C neoformans. It is important to suspect capsule-deficient C neoformans when the conventional diagnostic approaches fail to identify the organism or antigens.

Adult↗
PubMed1995

Binding of host collectins to the pathogenic yeast Cryptococcus neoformans: human surfactant protein D acts as an agglutinin for acapsular yeast cells.

Cryptococcus neoformans is an opportunistic pathogen in AIDS patients causing disseminated disease and lethal meningitis after inhalation of acapsular or sparsely encapsulated yeast cells. In this study we have investigated whether a recently described family of primitive opsonins, termed collectins, contribute to innate resistance against C. neoformans. The pulmonary surfactant proteins SP-A and SP-D as well as the serum collectins mannose-binding protein and CL-43 bound in a calcium-dependent manner to acapsular C. neoformans in vitro. Binding was concentration dependent and abolished by competition with defined mono- and oligosaccharides. In contrast, no binding of the collectins was observed with the encapsulated form of the yeast. Furthermore, binding of purified collectin SP-D, but not SP-A, mannose-binding protein, or CL-43, led to a concentration-dependent agglutination of acapsular C. neoformans. These data indicate that collectins recognize carbohydrate structures in the cell wall of an initial infectious form of C. neoformans and may play a role in early antifungal defenses in the lung.

Agglutinins↗