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PubMed2004

Molecular typing of Cryptococcus neoformans by PCR fingerprinting, in comparison with serotyping and Fourier transform infrared-spectroscopy-based phenotyping.

Molecular typing by PCR fingerprinting using the single primer (GACA)4 was performed with 110 isolates of Cryptococcus neoformans. Seventy clinical isolates of C. neoformans var. neoformans from Germany (n = 52) and Africa (n = 18) were included. Of these, serotype A (C. neoformans var. grubii) accounted for 47 isolates, serotype D for 12 and serotype AD for 11. Fourier transform infrared (FT-IR) spectroscopy was evaluated for its discriminatory power in phenotyping. Molecular types, defined by different PCR fingerprinting patterns, were compared to serotypes, and both sets of results were compared with the results of analysis by FT-IR spectroscopy. PCR fingerprinting revealed genotypic diversity within each serotype; it showed three different genotypes (designated VNA1-VNA3) within serotype A, two within serotype D (VND1 and VND2), and three within serotype AD (VNAD1-VNAD3). The nomenclature of molecular types within C. n. var. neoformans, as seen in publications to date, is not uniform. In this study, the name assigned to each genotype was based on the 98.6% concordance of genotypes with serotypes, a correspondence that facilitates interlaboratory comparison. This nomenclature is tentatively recommended as a standard. FT-IR spectroscopy combined with hierarchical cluster analysis successfully distinguished C n. var. neoformans from C. n. var. gattii. For C. n. var. neoformans, FT-IR confirmed three distinct genotypes within serotype A and was able to distinguish isolates derived from particular patients as well as isolates differing at the sub-genotype level. Within C. n. var. gattii, the serotypes B and C did not correlate with the four genotypes VGI-VGIV. However, these serotypes could clearly be separated by FT-IR spectroscopy. The molecular profiles were reproducible, and were more stable and more discriminating than serotyping. In connection with a standardized nomenclature, PCR fingerprinting can be a beneficial tool for global epidemiological studies. FT-IR spectroscopy adds an additional level of resolution.

Bacterial Typing Techniques↗
PubMed2004

Cryptococcus neoformans Kin1 protein kinase homologue, identified through a Caenorhabditis elegans screen, promotes virulence in mammals.

Cryptococcal infections are a global cause of significant morbidity and mortality. Recent studies support the hypothesis that virulence of Cryptococcus neoformans may have evolved via survival selection in environmental hosts, such as amoebae and free-living nematodes. We used killing of the nematode Caenorhabditis elegans by C. neoformans as an assay to screen a library of random C. neoformans insertion mutants. Of 350 mutants tested, seven were identified with attenuated virulence that persisted after crossing the mutation back into a wild-type strain. Genetic analysis of one strain revealed an insertion in a gene homologous to Saccharomyces cerevisiae KIN1, which encodes a serine/threonine protein kinase. C. neoformans kin1 mutants exhibited significant defects in virulence in murine inhalation and haematogenous infection models and displayed increased binding to alveolar and peritoneal macrophages. The kin1 mutant phenotypes were complemented by the wild-type KIN1 gene. These findings show that the C. neoformans Kin1 kinase homologue is required for full virulence in disparate hosts and that C. elegans can be used as a substitute host to identify novel factors involved in fungal pathogenesis in mammals.

Animals↗
PubMed1988

Benzoquinone activation of Cryptococcus neoformans capsular polysaccharide for construction of an immunoaffinity column.

p-benzoquinone was used as a two-step coupling reagent for preparation of an immunoaffinity absorbent in which the capsular polysaccharide of Cryptococcus neoformans was linked to an agarose gel. Cryptococcal polysaccharide is a difficult subject for chemical modification because it contains immunogenic O-acetyl groups which are sensitive to alkaline hydrolysis. The polysaccharide was activated by treatment with benzoquinone. The 'activated' polysaccharide was reactive with amino groups on AH-Sepharose. A pH of 8-9 was optimal for activation of the polysaccharide. Once activated, the polysaccharide was reactive with a model substrate, L-alanine-4-nitroanilide, over a pH range of 6-10. Since the O-acetyl groups are hydrolyzed at pH above 8.0, an activation pH of 8.0 and a coupling pH of 7.5 were used to prepare the conjugated gel. The polysaccharide immunoaffinity column was used successfully for isolation of rabbit antibodies to cryptococcal polysaccharide.

Benzoquinones↗
PubMed2004

In vitro activities of voriconazole, posaconazole, and fluconazole against 4,169 clinical isolates of Candida spp. and Cryptococcus neoformans collected during 2001 and 2002 in the ARTEMIS global antifungal surveillance program.

We examined the in vitro activities of voriconazole, posaconazole, and fluconazole against 3,932 isolates of Candida spp. and 237 isolates of Cryptococcus neoformans obtained from over 100 medical centers worldwide during 2001 and 2002. The MICs of the antifungal drugs were determined by broth microdilution tests performed according to the National Committee for Clinical Laboratory Standards (NCCLS) methods using RPMI 1640 as the test medium. Voriconazole and posaconazole were very active against Candida spp. (97-98% susceptible at MICs < or =1 microg/ml) and C. neoformans (98-100% susceptible at MICs < or =1 microg/mL). C. albicans (MIC90, 0.015-0.03 microg/ml) was the most susceptible species of Candida to both agents and C. glabrata (MIC90, 1-2 microg/mL) was the least susceptible. Both voriconazole and posaconazole were more active than fluconazole against all Candida spp. and C. neoformans. These results provide further evidence for the increased spectrum and potency of the new triazoles against a large and geographically diverse collection of opportunistic fungal pathogens.

Candida↗
PubMed2005

Killing of cryptococcus neoformans by Staphylococcus aureus: the role of cryptococcal capsular polysaccharide in the fungal-bacteria interaction.

Microbes compete for the environmental niche which is their host. To investigate the effects of a pathogenic bacterium on invasion and colonization by a pathogenic yeast, Cryptococcus neoformans was co-cultured with Staphylococcus aureus. We found that the number of colony forming units of C. neoformans was decreased by Staphylococcus aureus. In contrast, the viability of Candida albicans was not affected. Under the microscope, wild-type C. neoformans cells were shown to be surrounded by S. aureus, while cells of a capsuleless mutant of C. neoformans were not. C. neoformans was not killed when a membrane separated it from S. aureus in co-culture. Killing was confirmed by staining with cyanoditolyl tetrazolium chloride: S. aureus stained red, indicating viability, while C. neojormans did not stain, indicating lethality. The in situ terminal deoxynucleotidyl transferase-mediated dUTR nick end labeling (TUNEL) assay indicated cell death with fragmentation of DNA of C. neoformans. Capsular polysaccharide from C. neoformans inhibited the killing. Treatment of the crude polysaccharide with protease increased the inhibition. The protective activity resided in the glucuronoxylomannan (GXM) fraction, although the concentration required for the inhibition was high. These results suggest that S. aureus kills C. neoformans by a process that involves attachment to the cryptococcal capsule.

Cryptococcosis↗
PubMed2001

Susceptibilities of Cryptococcus neoformans strains to platelet binding in vivo and to the fungicidal activity of thrombin-induced platelet microbicidal proteins in vitro.

In this study we investigated the interactions between capsular and acapsular strains of Cryptococcus neoformans and blood platelets. In vivo microscopic observation of blood samples from mice inoculated with C. neoformans yeast cells demonstrated that encapsulated and nonencapsulated yeast cells disappeared quickly from the bloodstream and that platelets were attached solely to yeast cells of the nonencapsulated strains. In vitro we observed that only the acapsular strains were susceptible to the fungicidal activity of thrombin-induced platelet microbicidal proteins.

Animals↗
PubMed1998

Efficacy of NS-718, a novel lipid nanosphere-encapsulated amphotericin B, against Cryptococcus neoformans.

In vitro and in vivo efficacies of NS-718, a lipid nanosphere-encapsulated amphotericin B (AMPH-B), have been studied. Of the tested AMPH-B formulations, NS-718 had the lowest MIC for Cryptococcus neoformans. In a murine model, low-dose therapy (0.8 mg/kg of body weight) with NS-718 showed higher efficacy than that with AmBisome. High-dose therapy (2.0 mg/kg) with NS-718 was much more effective than those with Fungizone and AmBisome. In mice treated with a high dose of NS-718, only a few yeast cells had grown in lung by 7 days after inoculation. A pharmacokinetic study showed higher concentrations of AMPH-B in lung following administration of NS-718 than after administration of AmBisome. Our results indicated that NS-718, a new AMPH-B formulation, is a promising antifungal agent for treatment of pulmonary cryptococcosis and could be the most effective antifungal agent against C. neoformans infections.

Amphotericin B↗
PubMed1997

Macrophage inflammatory protein-1alpha (MIP-1alpha) is required for the efferent phase of pulmonary cell-mediated immunity to a Cryptococcus neoformans infection.

Our objective was to define the role of the chemokine macrophage-inflammatory protein-1alpha (MIP-1alpha) in the efferent phase of pulmonary cell-mediated immunity (CMI) against Cryptococcus neoformans. Following intratracheal inoculation of C. neoformans (24067) into CBA/J mice, the development of CMI was required for leukocyte recruitment into the lungs at 2 wk postinfection. MIP-1alpha mRNA was expressed by day 6 postinfection, and MIP-1alpha protein in bronchoalveolar lavage fluid was detectable at day 6, but significantly elevated at days 19 and 33. Administration of neutralizing anti-MIP-1alpha Abs from days 7 to 13 blocked the increase in bronchoalveolar lavage fluid MIP-1alpha and resulted in a 37% decrease in total leukocytes in the lungs at day 16. There were 66% fewer macrophages/monocytes and 42% fewer neutrophils in the lungs of anti-MIP-1alpha-treated mice, and the pulmonary burden of C. neoformans was threefold higher. There was no significant difference in the number of eosinophils, CD4+, CD8+, or B220+ lymphocytes between the two groups of mice. Neutralization of MIP-1alpha did not significantly decrease the levels of monocyte chemotactic protein-1 (MCP-1); however, neutralization of MCP-1 significantly decreased MIP-1alpha levels, demonstrating that induction of MIP-1alpha was largely dependent on MCP-1 production. Neutralization of MIP-1alpha also blocked the cellular recruitment phase of a recall response to cryptococcal Ag in the lungs of immunized mice. Thus, in both the contexts of active cryptococcal infection or rechallenge with cryptococcal Ag, MIP-1alpha was required during the efferent phase of CMI for maximal leukocyte recruitment into the lungs, most notably the recruitment of phagocytic effector cells (neutrophils and macrophages).

Animals↗
PubMed1999

Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages.

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2',7'-difluorofluorescein (Oregon Green). These probes have pKas of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 microM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.

Calibration↗
PubMed1979

Cryptococcus neoformans: gastronomic delight of a soil ameba.

During 7 days of incubation in vitro the trophozoite stage of the free-living soil amoeba, Acanthamoeba polyphaga, phagocytized and killed 78-97% of the cells of three strains of Cryptococcus neoformans. With one strain, incubation time was increased to nine days and 99% of the yeast cells were killed. It was calculated that during 4-9 days of incubation a single trophozoite phagocytized and killed a daily average of 84 yeast cells. The lethal effect of A. polyphaga on C. neoformans may represent a biological control mechanism in nature. Some of the surviving cells of C. neoformans developed into colonies containing pseudohyphae; these pseudolhyphal forms may be a biological 'escape hatch'.

Amoeba↗
PubMed2000

Cytokines and costimulatory molecules: positive and negative regulation of the immune response to Cryptococcus neoformans.

Cytokines are small proteins or glycoproteins that transmit information from one cell to another. Most cells in the body secrete and respond to cytokines and their effects have been described on a myriad of cellular functions. Cytokine interactions may not be linear, thus making the system extremely intricate and with unpredictable features. Therefore, each model of disease may be unique, with its own mechanism of autoregulation dictated by positive and negative feedback involving cytokines and costimulatory molecules. The emergence of some cytokines over others in the course of Cryptococcus neoformans infection may characterize a positive or negative outcome of cryptococcosis. Much less is known about the influence of costimulatory molecules in regulating C. neoformans immune response. The available information indicates a critical role for proinflammatory cytokines such as tumor necrosis factor alpha and interleukin 12 (IL-12). The positive role of interferon gamma in infected tissue as an inducer of antimicrobial function of innate immune cells and as positive feedback for IL-12 induction appears to be indisputable. In vitro studies indicate that costimulatory molecule expression appears to be regulated on antigen-presenting cells by C. neoformans and increased expression of B7-1 and CD40 on these cells may promote a protective response. These studies await confirmation in an in vivo system. The interplay between cytokines and costimulatory molecules has been scarcely explored and additional details are needed to better understand how they convey positive and negative information to immune cells in response to C. neoformans.

Antibody Formation↗
PubMed1999

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18.

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.

Animals↗
PubMed1991

[The defensive role of human pulmonary alveolar macrophages and polymorphonuclear leukocytes against a strongly or weakly virulent strains of Cryptococcus neoformans].

The phagocytic and killing activities of human pulmonary alveolar macrophages (PAM) and polymorphonuclear leukocytes (PMN), and superoxide (O2-) production in phagocytizing PAM and PMN were studied by using a strongly and weakly virulent strains of Cryptococcus neoformans (C. neoformans) in mice. The strongly virulent strain was encapsulated with a thick capsule while the other had a thin one. The phagocytic and killing activities of PAM and PMN against the strongly virulent strain were lower than against the weakly virulent one. There was no significant difference between PAM and PMN in terms of phagocytic or killing activities and O2- production. It seems likely that O2- plays no defensive role in the intracellular killing of C. neoformans by PAM and PMN because no correlation was found between the killing activity and O2- production. These results indicate that human PAM and PMN play an effective role in host defense against the weakly virulent strain of C. neoformans encapsulated with a thin capsule but are not effective against the strongly virulent strain encapsulated with a thick capsule.

Adolescent↗
PubMed2003

Role of alternative oxidase gene in pathogenesis of Cryptococcus neoformans.

We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37 degrees C in the human-pathogenic yeast Cryptococcus neoformans. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The C. neoformans alternative oxidase gene (AOX1) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An aox1 mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length AOX1. Compared to both the wild-type and reconstituted strains, the aox1 mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The aox1 mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The aox1 mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of C. neoformans can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.

Animals↗
PubMed2001

Role of mannoprotein in induction and regulation of immunity to Cryptococcus neoformans.

Our previous observations showed that mannoprotein (MP) induces early and massive production of interleukin-12 (IL-12) in vitro. This study was designed to investigate whether this phenomenon could be applied in vivo and to determine the biological significance of MP in Cryptococcus neoformans infection. The results reported here show that MP treatment induces IL-12 secretion by splenic macrophages and IL-12 p40 mRNA in the brain. During C. neoformans infection, MP reinforced IL-12 and IFN-gamma secretion that coincided with enhanced antifungal activity of natural effector cells, early resolution of the inflammatory process, and clearance of fungal load from the brain. These studies show that MP is a key inflammatory mediator that induces a protective immune response against C. neoformans infection. This information can be used to facilitate the design of a rational approach to manipulate the immune response to C. neoformans.

Animals↗
PubMed1997

Direct interactions of human natural killer cells with Cryptococcus neoformans inhibit granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha production.

Human natural killer (NK) cells and T lymphocytes can bind to and inhibit the growth of the yeast-like organism Cryptococcus neoformans. Binding of target cells to NK or T cells also has the potential to modulate cytokine production by the effector cells. In this study, we assessed the ability of C. neoformans to modulate NK cell production, or in some cases T-cell production, of granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor alpha (TNF-alpha). We found that freshly isolated human NK cells from most individuals make GM-CSF and TNF-alpha constitutively when cultured in vitro. The addition of C. neoformans to T-cell fractions which do not make GM-CSF constitutively did not affect GM-CSF production, but the addition of C. neoformans to NK cell fractions significantly reduced the amounts of GM-CSF produced in most NK cell samples. The reduction in the amount of GM-CSF in C. neoformans-NK cell cocultures could not be attributed to loss of lymphocyte viability or to C. neoformans adsorbing or degrading the cytokine and was dependent on direct contact between the NK cells and cryptococcal cells. GM-CSF was not the only cytokine to be down-regulated. TNF-alpha production was also diminished when NK cells were incubated with C. neoformans. The regulation of both cytokines was at the transcriptional level because GM-CSF and TNF-alpha mRNA levels were lower in NK cell samples incubated with C. neoformans than in NK cell samples incubated without C. neoformans. Diminished production of constitutively produced cytokines resulting from the interaction of NK cells with cryptococcal cells has the potential to affect phagocytic cells in the immediate regional environment and to damp the immune response.

Cryptococcus neoformans↗
PubMed2005

Thioredoxin reductase is essential for viability in the fungal pathogen Cryptococcus neoformans.

Thioredoxin reductase (TRR1) is an important component of the thioredoxin oxidative stress resistance pathway. Here we show that it is induced during oxidative and nitrosative stress and is preferentially localized to the mitochondria in Cryptococcus neoformans. The C. neoformans TRR1 gene encodes the low-molecular-weight isoform of the thioredoxin reductase enzyme, which shares little homology with that of its mammalian host. By replacing the endogenous TRR1 promoter with an inducible copper transporter promoter, we showed that Trr1 appears to be essential for viability of this pathogenic fungus, making it a potential antifungal target.

Cell Survival↗
PubMed2004

Clinical evaluation of a dried commercially prepared microdilution panel for antifungal susceptibility testing of five antifungal agents against Candida spp. and Cryptococcus neoformans.

A commercially prepared dried-broth microdilution panel (Sensititre, TREK Diagnostic Systems, Cleveland, OH) was compared with a reference frozen-broth microdilution panel for antifungal susceptibility testing of 728 clinical isolates of Candida spp. and 98 clinical isolates of Cryptococcus neoformans. The antifungal agents tested were amphotericin B, fluconazole, 5-fluorocytosine (5FC), itraconazole, and voriconazole. Microdilution testing was performed according to NCCLS recommendations. Minimum inhibitory concentration (MIC) endpoints were read visually after 48 hours of incubation (72 hours for C. neoformans) and were assessed independently for each microdilution panel. Discrepancies among MIC endpoints of no more than 2 log(2) dilutions were used to calculate the percentage of agreement. Overall levels of agreement between the study and reference panels were 98% for Candida spp. and 93% for C. neoformans. The agreement for each antifungal agent ranged from 96.6% for voriconazole to 99.4% for amphotericin B. The TREK dried microdilution panel appears to be a viable alternative to frozen-broth microdilution panels for testing of Candida spp. and C. neoformans.

Antifungal Agents↗