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PubMed1998

In vitro and in vivo efficacy of the triazole TAK-187 against Cryptococcus neoformans.

Multiple isolates of Cryptococcus neoformans, including those with fluconazole resistance, were tested to assess the in vitro activity of the new triazole TAK-187. MICs of TAK-187 were at least eightfold lower than those of fluconazole, and fungicidal concentrations for most isolates were 4 microg/ml or less. TAK-187 also was evaluated as intermittent therapy using two dosages in a rabbit model of experimental cryptococcal meningitis. Compared to daily treatment with fluconazole, as little as two doses of TAK-187 given 7 days apart were found to be effective. Plasma and cerebrospinal fluid TAK-187 concentrations were many times higher than MICs and fungicidal concentrations. Based upon its therapeutic efficacy and long half-life in the rabbit model, TAK-187 should be investigated for intermittent dosing in treatment or suppression of cryptococcal infections in humans.

Animals↗
PubMed2001

Spatial and temporal sequence of capsule construction in Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans is distinguished by an extensive polysaccharide capsule, which impedes host defences and is absolutely required for fungal virulence. Despite the biological importance of the capsule, nothing is known about how it is assembled. Substantial capsule growth occurs in two distinct situations relevant to cryptococcal pathogenesis: formation of new buds and induction of capsule on mature cells. We developed pulse-chase protocols to examine these events in a dynamic way using a variety of microscopy techniques. We show that the capsule overlying buds is newly synthesized and differs physically from the corresponding parental material. New capsule formed by mature cells upon induction of synthesis is added at the inner aspect of the existing structure, displacing pre-existing material outwards. Surprisingly, new polysaccharide material is also deposited throughout the capsule, yielding a progressively denser structure. These results yield the first model of capsule synthesis and open new lines of investigation into the underlying mechanisms.

Cell Wall↗
PubMed1994

Synergic inhibitory activity of amphotericin-B and gamma interferon against intracellular Cryptococcus neoformans in murine macrophages.

Cryptococcus neoformans is responsible for pulmonary and meningal infections in HIV patients. The lack of effective cellular cooperation caused by the low level of CD4+ cells, and the resistance of C. neoformans to phagocytosis allows growth and persistence of the yeast in the host. We describe here an in-vitro model of intracellular replication of C. neoformans inside J774-A.1 macrophages, and the determination of the intracellular antifungal activity of amphotericin B and fluconazole alone or in association with IFN-gamma. The maximum inhibitory effect was observed with one MIC of amphotericin B and 100 or 1000 IU/mL of IFN-gamma. amphotericin B alone (at 1 x MIC), or either 1 x or 50 x MIC of fluconazole in normal or IFN-gamma activated macrophages, did not eradicate the ingested yeast. A potential underlying mechanism of the synergy of amphotericin B in IFN-gamma primed macrophages was investigated by measurement of nitrite level and by use of the NO synthase competitive inhibitor, NG-monomethyl L-arginine (NMMA). One MIC of amphotericin B was able to activate the synthesis of nitrogen reactive intermediates in IFN gamma-primed macrophages. NMMA treated infected macrophages responded less well to IFN-gamma priming, resulting in a moderate inhibition in subsequent amphotericin B exposure.

Amphotericin B↗
PubMed2006

Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.

Cryptococcus neoformans (Cn) is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease. The interaction of Cn with alveolar macrophages is critical for containing the infection , but Cn can also replicate intracellularly and lyse macrophages . Cn has a unique intracellular pathogenic strategy that involves cytoplasmic accumulation of polysaccharide-containing vesicles and intracellular replication leading to the formation of spacious phagosomes in which multiple cryptococcal cells are present . The Cn intracellular pathogenic strategy in macrophages and amoebas is similar, leading to the proposal that it originated as a mechanism for survival against phagocytic predators in the environment . Here, we report that under certain conditions, including phagosomal maturation, possible actin depolymerization, and homotypic phagosome fusion, Cn can exit the macrophage host through an extrusion of the phagosome, while both the released pathogen and host remain alive and able to propagate. The phenomenon of "phagosomal extrusion" indicates the existence of a previously unrecognized mechanism whereby a fungal pathogen can escape the intracellular confines of mammalian macrophages to continue propagation and, possibly, dissemination.

Actins↗
PubMed1997

T cells cooperate with passive antibody to modify Cryptococcus neoformans infection in mice.

Cryptococcus neoformans is an encapsulated fungus that is a major cause of meningitis in patients with AIDS. In immunocompetent mice, administration of IgG1 mAb protects against cryptococcal infection, whereas administration of IgG3 is not protective and can accelerate the infection. In beige mice with impaired natural killer cell function, the effects of IgG1 and IgG3 are similar to those observed in immunocompetent mice, suggesting that natural killer cells are not crucial for antibody-mediated modulation of cryptococcal infection. In mice lacking CD4+ T cells, IgG1 is not protective and IgG3 accelerates infection, indicating that CD4+ T cells are required for antibody-mediated protection. In mice lacking CD8+ T cells, both IgG1 and IgG3 antibodies prolong survival, indicating that acceleration of the disease process by IgG3 involves CD8+ T cells. Both IgG1-mediated protection and IgG3-mediated acceleration of infection require interferon gamma. These results reveal a functional dependence of passively administered antibody on cellular immunity in cryptococcal infection in mice and have implications for antibody-based therapies in humans in the setting of CD4+ lymphopenia.

Animals↗
PubMed1991

Early events in initiation of alternative complement pathway activation by the capsule of Cryptococcus neoformans.

The capsule of Cryptococcus neoformans is a powerful activator of the alternative complement pathway. This study examined the manner in which the cryptococcal capsule influences initiation of and early events in complement activation by C. neoformans. These studies examined the effects of the classical and alternative pathways on the kinetics and early sites for deposition of C3 fragments on encapsulated cryptococci, nonencapsulated cryptococci, and zymosan. The results showed that nonencapsulated cryptococci and zymosan are qualitatively and quantitatively similar in the manner in which they initiate complement activation. Both utilize the classical and alternative pathways. Initiation via the classical pathway occurs suddenly and simultaneously at sites distributed over the entire cell surface. Initiation of the alternative pathway by zymosan and nonencapsulated cryptococci is characterized by a lag of 6 to 8 min before appreciable amounts of C3 accumulate on the cells. Alternative pathway initiation by zymosan and nonencapsulated cryptococci occurs at a limited number of focal initiation sites that expand with alternative pathway amplification to cover the cell surface. Presence of the cryptococcal capsule blocks classical pathway initiation, which would normally occur at the cryptococcal cell wall, and produces an initiation that is dependent solely on the alternative pathway. Initiation of the alternative pathway by the cryptococcal capsule is characterized by a lag in C3 accumulation and the appearance of a limited number of focal initiation sites which resemble those observed when the alternative pathway is activated by zymosan and nonencapsulated cryptococci.

Complement Activation↗
PubMed2002

Physical maps for genome analysis of serotype A and D strains of the fungal pathogen Cryptococcus neoformans.

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies

Chromosomes, Artificial, Bacterial↗
PubMed1997

Ferric iron reduction by Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans must reduce Fe(III) to Fe(II) prior to uptake. We investigated mechanisms of reduction using the chromogenic ferrous chelator bathophenanthroline disulfonate. Iron-depleted cells reduced 57 nmol of Fe(III) per 10(6) cells per h, while iron-replete cells reduced only 8 nmol of Fe(III). Exponential-phase cells reduced the most and stationary-phase cells reduced the least Fe(III), independent of iron status. Supernatants from iron-depleted cells reduced up to 2 nmol of Fe(III) per 10(6) cells per h, while supernatants from iron-replete cells reduced 0.5 nmol of Fe(III), implying regulation of the secreted reductant(s). One such reductant is 3-hydroxyanthranilic acid (3HAA), which was found at concentrations up to 29 microM in iron-depleted cultures but <2 microM in cultures supplemented with iron. Moreover, when washed and resuspended in low iron medium, iron-depleted cells secreted 20.4 microM 3HAA, while iron-replete cells secreted only 4.5 microM 3HAA. Each mole of 3HAA reduced 3 mol of Fe(III), and increasing 3HAA concentrations correlated with increasing reducing activity of supernatants; however, 3HAA accounted for only half of the supernatant's reducing activity, indicating the presence of additional reductants. Finally, we found that melanized stationary-phase cells reduced 2 nmol of Fe(III) per 10(6) cells per h--16 times the rate of nonmelanized cells--suggesting that this redox polymer participates in reduction of Fe(III).

3-Hydroxyanthranilic Acid↗
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↗
PubMed2001

[Construction of Cryptococcus neoformans cap70 transformation system].

OBJECTIVE: To construct a transformation system of Cryptococcus neoformans capsule-deficient strain cap70. METHODS: (1)Obtained ura5 mutants by screening the Cryptococcus neoformans cap70 using 5-fluoroorotic acid counter selection method, analyzed the obtaining strain by Southern blot and PCR; (2)Through the plasmid pCXJ18 and pCXJU which contained ura5 gene from Cryptococcus neoformans and ura3 from Kluyveromyces fragilils. as a selective marker, the ura5 strains were transformed by electrotransformation and chemical transformation. RESULTS: Obtained ura5 mutants, they were detected as lack of ura function. The chemical transformation method was first be used in Cryptococcus neoformans transformation. CONCLUSION: A transformation system of Cryptococcus neoformans cap70 has been established, which provides not only the basis for gene cloning from the species for studying gene function and gene expression.

Cryptococcus neoformans↗
PubMed1987

Failure of the Bactec 460 radiometer to detect Cryptococcus neoformans fungemia in an AIDS patient.

Cryptococcus neoformans fungemia occurred in a patient with the acquired immunodeficiency syndrome (AIDS). The BACTEC 460 radiometer failed to detect Cryptococcus neoformans in eight aerobic BACTEC 6B culture bottles inoculated with the patient's blood. The diagnosis of cryptococcemia was established by terminal (seven-day) subculturing of 6B broth to chocolate agar, which was positive for all eight radiometrically negative blood culture bottles. It appears that radiometric measurement is not optimal for the laboratory detection of cryptococcal fungemia.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis.

Cryptococcus neoformans var. neoformans is an important opportunistic fungal pathogen of patients whose immune system has been compromised due to viral infection, antineoplastic chemotherapy, or tissue transplantation. As many as 13% of all AIDS patients suffer a life-threatening cryptococcal infection at some time during the course of their HIV disease. To begin to understand the molecular basis for virulence in Cryptococcus neoformans var. neoformans serotype A, we have employed signature-tagged mutagenesis (STM) to identify mutants with altered virulence in a mouse model. The critical parameters of signature-tagged mutagenesis in C. neoformans are explored. Data are presented showing that at least 100 different strains can be mixed together in a single animal with each participating in the infection and that there is no apparent interaction between a virulent strain and an avirulent strain in our animal model. Using signature-tagged mutagenesis, we identified 39 mutants with significantly altered growth in a competitive assay. Molecular analyses of these mutants indicated that 19 (49%) contained an insertion in the actin promoter by homologous recombination from a single crossover event, creating a duplication of the actin promoter and the integration of single or multiple copies of the vector. Analysis of the chromosomal insertion sites of those mutants that did not have an integration event in the actin promoter revealed an approximately random distribution among the chromosomes. Individual challenge of the putative mutants in a mouse model revealed five hypovirulent mutants and one hypervirulent mutant.

Actins↗
PubMed1986

Proteolytic activity of a clinical isolate of Cryptococcus neoformans.

A clinical isolate of Cryptococcus neoformans was grown on essential salts medium without (NH4)2SO4 and supplemented with bovine serum albumin as the sole carbon and nitrogen source. Growth on this medium resulted in secretion of proteolytic enzymes by the organism. The secretion of protease enzymes was inhibited by (NH4)2SO4 and glucose. Proteolytic activity may be a factor in virulence and pathogenicity of C. neoformans.

Cryptococcus↗
PubMed1995

Microcomputer system for automatic identification of the Cryptococcus neoformans and its clinical application.

In this study, microcomputer image processing and pattern recognition technology, and the knowledge of morphology and optical characteristics of Cryptococcus neoformans were used for identification of Cryptococcus neoformans. Four groups of mice were lethally infected with standard strain, Wuhan strain, American B-2643 strain and Var. Shanghainesis of the Cryptococcus neoformans. The samples collected included mice brain, lung, kidney, liver, small intestine tissue and were observed under a light microscope. More than 600 images of the fungus were input into a microcomputer. A system of computer for automatic identification of the Cryptococcus neoformans was developed. The technique involved image preprocessing, image segmenting, coding of line-length on the edge, curve fitting, extracting of image feature, building of image library and feature data bank etc.. And then, 768 images of the clinical samples and other fungus samples whose morphological features tend to be confused with Cryptococcus neoformans were input into microcomputer and subjected to automatic identification. The Cryptococcus neoformans was accurately identified within 15 min, and the consistency rate with results of routine culture was 98%.

Animals↗
PubMed2003

Phenotyping of Cryptococcus neoformans strains in Bergamo, Italy (1985-2000).

Cryptococcus neoformans is the cause of the most common life-threatening fungal infection in patients with AIDS. Thirty strains of C. neoformans were collected from inpatients and typied evaluating activity, morphotyping, serotyping, chemosensitivity and adhesivity. Cryptococcus neoformans strains showed different aspectotype profile, the sole presence of serotypes A and D, good susceptibility to azoles and Amphotericin B. Phenotypic epidemiologic markers can be used: characterization of clinical strains excludes a common source.

Adult↗
PubMed1986

Encapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans.

Acapsular (Cap-) mutants of Cryptococcus neoformans var. neoformans that produce melanin (Mel+) on diphenol media at 30 degrees C but not at 37 degrees C were found to be avirulent for mice. Compared with wild-type isolates, the mutants had a lower rate of L-3,4-dihydroxyphenylalanine uptake at 37 degrees C and showed an insignificant level of phenoloxidase activity at both temperatures. To study the relationship of Cap and Mel phenotypes to virulence in mice, we crossed one of the mutants (Cap- Mel-) with a wild type (Cap+ Mel+) to obtain four classes of progeny (Cap+ Mel+, Cap+ Mel-, Cap- Mel+, and Cap- Mel-). The progeny with the Cap+ Mel+ phenotype and the wild-type parent (Cap+ Mel+) were inoculated into mice (10(6) cells per mouse) and, within 40 days, produced fatal infection in 90 to 100% of the animals. None of the other three phenotypes produced fatal infection within the same period. While progeny with the Cap+ Mel- phenotype did produce fatal infection after 40 days, 70 to 90% of the mice survived at least until day 70. However, in the isolates recovered from the brain tissue of a mouse that died on day 68, nearly 40% of the CFU had reverted to the Cap+ Mel+ type. The virulence of one of these revertant Cap+ Mel+ isolates was compared with that of a Cap+ Mel- isolate recovered from the same tissue. One hundred percent of the mice inoculated with the revertant died within 35 days, while no fatal infection was produced in the mice inoculated with the Cap+ Mel- isolate within the same period. The isolates with the Cap- Mel+ or Cap- Mel- phenotype not only failed to produce fatal infection but failed to revert to the Cap+ Mel+ type in the mouse brain during the experimental period. These results indicate that both the Cap+ phenotype and the Mel+ phenotype are important indicators of virulence in C. neoformans.

Animals↗
PubMed1997

Laccase and melanin in the pathogenesis of Cryptococcus neoformans.

Cryptococcosis, caused by an encapsulated fungus, Cryptococcus neoformans, has emerged as a life threatening infection in HIV positive individuals and other immunocompromised hosts. The present review describes laccase and its product melanin as an important virulence factor of Cryptococcus neoformans and illustrates the approaches used in elucidating the pathogenesis of cryptococcosis. Characterization of the biochemical pathways leading to melanin synthesis is summarized using biochemical and biomolecular approaches. Melanin synthesis is dependent on a single copper-dependent enzyme, laccase. Since the mammalian host does not contain this enzyme, laccase is an attractive candidate for the study of fungal pathogenesis, as well as a drug target. The cloning of the CNLAC1 gene and construction of CNLAC1 gene knock-out strains has confirmed its role in the virulence of Cryptococcus. Also described is the role of melanin in the host-pathogen interactions. Melanin may protect Cryptococcus cells by a variety of methods including anti-oxidant or cell wall surface effects thereby offering protection against numerous effectors of cellular immunity.

Antioxidants↗