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PubMed1995

Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry.

Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2'-7(1)-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% +/- 2%, 35% +/- 14%, 50% +/- 11%, 51% +/- 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% +/- 17%, 22% +/- 9%, and 40% +/- 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.

Blood Bactericidal Activity↗
PubMed1990

Pathogenesis of Cryptococcus neoformans in congenitally immunodeficient beige athymic mice.

Mortality after intravenous challenge with 10(4) Cryptococcus neoformans demonstrated that doubly immunodeficient beige athymic (bg/bg nu/nu) mice were more susceptible to systemic cryptococcosis than either bg/bg or nu/nu mice. Infected bg/bg nu/nu mice also had a shortened lifespan compared with their bg/bg nu/+ littermates. Beige athymic (bg/bg nu/nu) but not bg/bg nu/+mice developed cryptococcal lesions in the skin, demonstrating that C. neoformans is dermatotropic in a T-cell-deficient host. Higher numbers of C. neoformans were isolated from the lungs and spleen of infected bg/bg nu/nu than bg/bg nu/+ mice as early as day 3 after challenge, indicating that in lymphoid-rich organs, T cells can alter the course of systemic cryptococcosis early in the infection. Despite extensive abscess formation in the brains of bg/bg nu/+ mice, dissemination and growth rate of C. neoformans in the brain was similar in both genotypes. The primary histopathological feature in tissues from bg/bg nu/nu mice infected with C. neoformans consisted of foci of encapsulated yeast cells with minimal to no inflammatory response. In contrast to bg/bg nu/nu mice, bg/bg nu/+ mice mounted a vigorous inflammatory response to C. neoformans that progressed from acute to chronic inflammation. Beige athymic mice are a new animal model that will be useful in clarifying the innate and acquired immune factors important in resistance to cryptococcosis.

Animals↗
PubMed1992

Experimental model of intracerebral infection with Cryptococcus neoformans: roles of phagocytes and opsonization.

A murine model of intracerebral (i.c.) infection with Cryptococcus neoformans in which naive mice receiving an i.c. fungal inoculation developed a severe disease has been established. The effect was strictly dependent on the number of microorganisms injected and evolved as lethal meningoencephalitis. Murine susceptibility to i.c. infection with C. neoformans was enhanced by treatment with chloroquine and colchicine, agents known to greatly affect the host phagocytic compartment. Furthermore, the life spans of both naive and drug-treated mice were significantly augmented when opsonized fungi were injected. Therefore, phagocyte-mediated mechanisms are likely involved in local resistance to i.c. infection with C. neoformans. Further support for this conclusion was supplied by in vitro data showing that microglial cells were proficient anticryptococcal effectors, provided opsonized microorganisms were used.

Animals↗
PubMed2000

Do kinetics of the humoral response to Cryptococcus neoformans proteins during murine cryptococcosis reflect outcome?

The kinetics of the humoral response to Cryptococcus neoformans proteins were studied in outbred mice infected with isolate NIH52D. Future nonsurvivors had earlier and stronger (i.e., more bands recognized) humoral responses than survivors. In addition, antibodies to a 56- to 60-kDa membrane antigen and to a 39- to 40-kDa cytosolic antigen were detected more frequently in samples from future nonsurvivors and from survivors, respectively (P < 0.05).

Animals↗
PubMed1995

Clinical and host differences between infections with the two varieties of Cryptococcus neoformans.

A population-based register of cases of cryptococcosis in patients treated in Victoria, Australia, over a 10-year period was established for studying the epidemiologic and clinical features of infection with Cryptococcus neoformans and its two varieties, gattii and neoformans. One hundred thirty-three cases of cryptococcosis were entered on the register; the incidence was 3.0 cases per 1 million population per year, a rate that increased to 5.0 cases per 1 million population per year over the decade as a result of the AIDS epidemic. There was a distinct association between immune status and C. neoformans variety: all C. neoformans variety gattii infections occurred in healthy hosts and 90% of C. neoformans variety neoformans infections occurred in immunosuppressed hosts. Meningitis was the commonest manifestation, with focal CNS and pulmonary lesions occurring primarily in healthy hosts with C. neoformans variety gattii infection; isolation of C. neoformans from blood and urine was associated with immunosuppression and C. neoformans variety neoformans infection. The mortality among patients with C. neoformans variety neoformans infection was high, while none of those patients with C. neoformans variety gattii died but often had neurological sequelae that required surgery and prolonged therapy. These findings appear to be related to variety-specific interactions between host and parasite and warrant further epidemiologic and immunologic study.

Adolescent↗
PubMed1999

Cryptococcus neoformans differential gene expression detected in vitro and in vivo with green fluorescent protein.

Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungus Cryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFalpha1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFalpha1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFalpha1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFalpha1 promoter is induced by nutrient deprivation and the MATalpha locus containing the MFalpha1 gene has been linked with virulence, yeast cells containing the pMFalpha1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFalpha1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFalpha1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

Actins↗
PubMed2006

Molecular epidemiology of clinical and environmental isolates of the Cryptococcus neoformans species complex reveals a high genetic diversity and the presence of the molecular type VGII mating type a in Colombia.

The aim of this study was to investigate the epidemiological relationships of clinical and environmental isolates of the Cryptococcus neoformans species complex in Colombia. The current study reflects data from 1987 to 2004. In Colombia serotypes A and B are most frequently recovered from patients and the environment. Of the 178 clinical isolates studied, 91.1% were of serotype A, 8.4% serotype B and 0.5% serotype C. Of the 247 environmental isolates, 44.2% were of serotype A, 42.6% serotype B and 13.2% serotype C. No serotype D isolates were isolated. Serotype AD has not been recovered in Colombia. PCR fingerprinting with the primers M13, (GACA)4 and (GTG)5 and URA5 gene restriction fragment length polymorphism analysis grouped the majority of clinical serotype A and environmental serotype B isolates into the molecular types VNI (98.1%) and VGII (100%), respectively. Mating type alpha was determined in 99.3% of serotype A isolates, but 96.6% of serotype B isolates were of mating type a. Similar profiles between clinical and environmental isolates suggest that the patients may have acquired the infection from the environment. The data presented form part of the Colombian contribution to the ongoing global survey of the C. neoformans species complex.

Animals↗
PubMed1999

Differential regulation of immune responses by highly and weakly virulent Cryptococcus neoformans isolates.

Early inflammatory responses, delayed-type hypersensitivity (DTH) responses, and cytokine profiles were studied in mice infected by the pulmonary route with either a highly virulent isolate (NU-2) or a weakly virulent isolate (184A) of Cryptococcus neoformans. After infection, NU-2 remained in the lungs and the capsule became more pronounced during the first 24 h, whereas 184A induced an immediate inflammatory reaction and was rapidly cleared from the lungs. Cryptococcal antigen (GXM) appeared in sera early after infection with NU-2 and increased over the entire observation period. There was no detectable GXM in sera from 184A-infected mice. Both C. neoformans isolates induced anticryptococcal cell-mediated immune responses, but the responses had different profiles. DTH in NU-2-infected mice appeared at day 15 after infection and waned by day 21, whereas DTH in 184A-infected mice was present by day 5 and continued to increase. T helper 1 (Th1) cytokines (interleukin 2 [IL-2] and gamma interferon) were made by spleen cells early after infection with either isolate. NU-2-infected mice lost their ability to produce these cytokines, but 184A-infected mice retained it. IL-4, a Th2 cytokine, was not detected in infected mice. The regulatory cytokine IL-10 was made by spleen cells early but not later after infection with the highly virulent isolate and was not produced by spleen cells from 184A-infected mice. IL-10-deficient mice survived an NU-2 infection significantly longer than wild-type mice, suggesting that IL-10 is important in down-regulating the protective immune response. The induction of anergy appears to be responsible for the inability of NU-2-infected mice to control a C. neoformans infection.

Acute-Phase Reaction↗
PubMed2000

Quantitative analysis of phagocytosis of Cryptococcus neoformans by adherent phagocytic cells by fluorescence multi-well plate reader.

Macrophages and monocytes are adherent phagocytic cells which play an important role in host defence against the yeast-like fungus Cryptococcus neoformans. Before, phagocytosis by adherent phagocytes could only be measured by means of microscopy or by a radioactive assay, which both have obvious disadvantages. We have developed a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent phagocytes (e.g. alveolar macrophages) using a fluorescence multi-well plate reader. This method allows us to discriminate accurately between adherence and internalisation of C. neoformans by macrophages during long term incubation. In addition, the method was used to study the role of the mannose receptor in phagocytosis of the acapsular yeast in the absence of serum by human monocyte-derived macrophages (MDM).

Cell Adhesion↗
PubMed2006

Towards a vaccine for Cryptococcus neoformans: principles and caveats.

In the Damage-response framework of microbial pathogenesis, infectious diseases are one outcome of a host-microorganism interaction in a susceptible host. In cryptococcal disease, damage to the host is caused by Cryptococcus neoformans virulence determinants, the nature of the host response, or both. Further, the disease may be acute or reactivated from a latent state. Hence, a vaccine for C. neoformans would need to prevent disease resulting from either acute or reactivated infection. The evidence to support the development of a vaccine for C. neoformans that induces antibody-mediated immunity is discussed herein.

Animals↗
PubMed1996

gamma Interferon gene expression and release in human lymphocytes directly activated by Cryptococcus neoformans and Candida albicans.

Previous studies in our laboratory and others have demonstrated that T and/or NK cells can directly bind to and inhibit the growth of the medically important fungal pathogens Cryptococcus neoformans and Candida albicans by apparently non-major histocompatibility complex-restricted mechanisms. Here, we examined whether this direct interaction between lymphocytes and fungi also results in cytokine gene expression and release. Nonadherent lymphocytes (NAL), isolated from human peripheral blood mononuclear cells by depletion of cells adherent to plastic and nylon wool, released gamma interferon (IFN-gamma), but not interleukin-4 (IL-4) and IL-10, following stimulation with C. neoformans yeast cells and C. albicans yeast cells, hyphae, and supernatants. The fungal stimuli also induced IFN-gamma mRNA, with peak gene expression seen at or after 18 h. IFN-gamma release was still seen even when either NK cells or T lymphocytes were depleted by negative selection, suggesting that both cell types can be stimulated by fungi to produce IFN-gamma. Release of IFN-gamma from fungus-stimulated NAL occurred in the absence of an intact complement system and was not especially enhanced by culture with IL-2 or IL-12. These data expand the mechanisms by which the direct interaction of NAL with fungal targets can lead to immune activation. Moreover, to our knowledge, this is the first demonstration of direct stimulation of T-cell cytokine release by microbial pathogens.

Candida albicans↗
PubMed2003

'Ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi--the Cryptococcus neoformans paradigm.

Environmental pathogenic fungi present a paradox in that they are virulent in animals without requiring animal hosts for replication or survival, a phenomenon we call 'ready-made' virulence. In the human pathogenic fungus Cryptococcus neoformans, the capacity for virulence in animals may originate from environmental selective pressures imposed by such organisms as amoeboid and nematode predators. Many C. neoformans virulence factors appear to have 'dual use' capabilities that confer survival advantages in both animal hosts and in the environment. The findings with C. neoformans may provide a paradigm for understanding the origin and maintenance of virulence in other pathogenic environmental fungi.

Animals↗
PubMed1997

Molecular epidemiology of Cryptococcus neoformans in Brazil and the United States: evidence for both local genetic differences and a global clonal population structure.

Little is known about the global molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans. We studied 51 clinical and environmental (pigeon excreta) isolates from two cities in Brazil (Belo Horizonte and Rio de Janeiro) by analyzing their carbon assimilation patterns, electrophoretic karyotypes, restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element-1 (CNRE-1), and URA5 sequences. Results were compared to those previously obtained for isolates from New York City by the same DNA typing methods. Computer-assisted analysis of RFLPs and contour-clamped homogeneous electrophoresis (CHEF) patterns and URA5 sequences was performed to generate dendrograms. Some environmental and clinical isolates were found to be indistinguishable by CHEF, CNRE-1 RFLP, and URA5 sequence analyses. Similarly, some isolates from Rio de Janeiro and Belo Horizonte were indistinguishable by the three DNA typing techniques. Overall, Brazilian isolates appeared to be less heterogeneous by DNA analysis than isolates from other regions. Several Brazilian isolates were highly related to New York City isolates. Phylogenetic analysis of the sequences obtained for the Brazilian isolates and those obtained for New York City isolates was congruent with the dendrogram generated from the CNRE-1 RFLP data. In summary our results indicate (i) that the discriminatory power of the DNA typing method differs for Brazilian and New York City strains, with the order being CNRE-1 RFLP analysis > URA5 sequence analysis > CHEF analysis and CHEF analysis > URA5 sequence analysis > CNRE-1 RFLP analysis, respectively; (ii) that there are differences in local genetic diversity for Brazilian and New York City isolates; (iii) that there is additional evidence linking clinical isolates to those in pigeon excreta; and (iv) that some isolates from Brazil and New York City are closely related, consistent with the global dispersal of certain pathogenic strains.

Animals↗
PubMed1997

Human salivary histatin-5 exerts potent fungicidal activity against Cryptococcus neoformans.

Human salivary histatins (Hsns) have been known to be potent antifungal agents against Candida albicans for more than a decade. Here, we report that Hsns are also effective in killing another medically important opportunistic fungal pathogen, Cryptococcus neoformans, which has become a new threat among the immunocompromised patients, especially AIDS patients. In fact, the cidal activity of Hsn-5 against C. neoformans is as high as that against C. albicans.

Amino Acid Sequence↗
PubMed2006

Comparison of three commercial assays and a modified disk diffusion assay with two broth microdilution reference assays for testing zygomycetes, Aspergillus spp., Candida spp., and Cryptococcus neoformans with posaconazole and amphotericin B.

We compared posaconazole M27-A2 and M38-A MICs to Etest and YeastOne MICs for 92 zygomycetes, 126 Aspergillus isolates, 110 Candida isolates, and Cryptococcus neoformans. Reference MICs were also correlated with inhibition zone diameters in millimeters (modified M44-A disk and Neo-Sensitabs tablet methods). Etest MICs were obtained on solidified (1.5% agar) RPMI 1640 (2% dextrose), and zone diameters were obtained on supplemented (2% glucose and 0.5 microg/ml methylene blue [for all isolates]) and nonsupplemented Mueller-Hinton (MH; molds only) agar. MICs and zone diameters were obtained between 16 and 72 h. The overall agreement (% MIC pairs within a three-dilution range) between reference posaconazole and YeastOne MICs was 98 to 100% at 16 to 24 h for zygomycetes and yeasts and 99% at 24 to 48 h for Aspergillus. The overall agreement was lower between reference posaconazole and Etest MICs (94 to 97%) and by both methods with amphotericin B for all species (95 to 99.3%). For yeasts, the correlation coefficient was similar between reference posaconazole MICs and either disk (R, 0.810) or tablet (R, 0.769) zone diameter at 24 h and was superior on MH agar for molds at 16 to 48 h (R, 0.804 and 0.799 for disk and tablet, respectively). For amphotericin B, the best correlation between reference MICs and zone diameters was observed at 16 to 48 h for molds on MH agar (R, 0.736 to 0.812 and 0.765 to 0.749 for disk and tablet, respectively) and at 48 h for yeasts (R, 0.681 and 0.503 for disk and tablet, respectively). These data suggest the potential value of these alternative broth dilution and agar diffusion methods for testing posaconazole and amphotericin B in the clinical laboratory against the species evaluated.

Amphotericin B↗
PubMed1991

Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin.

We studied the receptors on human cultured macrophages (MO-M phi) responsible for binding encapsulated and isogenic mutant acapsular strains of Cryptococcus neoformans, and whether such binding leads to a phagocytic event. Both strains required opsonization with complement components in normal human serum in order for binding to occur. Binding of the acapsular, but not the encapsulated, strain led to phagocytosis. MAb directed against any of the three defined complement receptors (CR) on MO-M phi (CR1, CR3, and CR4) profoundly inhibited binding of serum-opsonized encapsulated (and to a lesser extent acapsular) organisms to MO-M phi. Immunofluorescence studies demonstrated migration of CR to the area of the cryptococcal binding site. Trypsin and elastase inhibited binding of encapsulated and, to a lesser extent, acapsular yeasts to MO-M phi. Binding of encapsulated C. neoformans was profoundly inhibited by incubation in the cold or by inhibitors of receptor capping and actin microfilaments. Thus, multiple CR appear to contribute to binding of serum-opsonized encapsulated C. neoformans by MO-M phi. Binding is an energy-dependent process that requires conformational changes in actin yet does not lead to phagocytosis of the organism. In contrast, energy is not required for binding of acapsular yeasts by MO-M phi and binding triggers phagocytosis.

Actins↗
PubMed2007

The mating type-specific homeodomain genes SXI1 alpha and SXI2a coordinately control uniparental mitochondrial inheritance in Cryptococcus neoformans.

In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type alpha (MATalpha) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type alpha (MATalpha)-specific gene SXI1a, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATalpha parent, and both the MATa and the MATalpha parents. In addition, progeny from same-sex mating between MATalpha strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1alpha and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.

Cryptococcus neoformans↗