Primary isolation medium for Cryptococcus neoformans.
The isolation and identification of Cryptococcus neoformans is improved by use of a potato dextrose medium containing urea-antibiotic supplements and a pH of 3.5.
Bacteria↗
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Pigment production of Cryptococcus neoformans grown with extracts of Guizotia abyssinica.
Brown pigment(s) formed in Cryptococcus neoformans when grown on media containing extracts of the seeds of Guizotia abyssinica cannot be extracted by common organic solvents or by 6 n HCl or 2 n NaOH. A similar pigmentation was observed in C. neoformans when grown on a medium containing caffeic acid isolated from the hydrolyzed methanol extract of G. abyssinica seeds. Its methyl ester and the diacetate thereof, as well as the following structurally related compounds, 3-hydroxytyramine, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylethanolamine, and 4-hydroxy-3,5-dimethoxycinnamic acid, brought about similar pigmentation. However, 2,4-, 2,5-, 2,6-, and 3,5-dihydroxybenzoic acids, tyrosine, phenylalanine, cinnamic acid, 4-hydroxycinnamic acid, and 4-hydroxy-3-methoxycinnamic acid did not cause coloration in C. neoformans.
Cinnamates↗
Prevalence in Cryptococcus neoformans strains of a polysaccharide epitope which can elicit protective antibodies.
Monoclonal antibody (MAb) 2H1 binds to an epitope in the capsule of Cryptococcus neoformans that can elicit protective antibodies. The binding of MAb 2H1 to C. neoformans strains was studied by agglutination, immunofluorescence, and phagocytosis assays. The MAb 2H1 epitope was present in all 21 isolates studied, including those recovered from patients with recurrent infections.
Agglutination↗
Contribution of epitope specificity to the binding of monoclonal antibodies to the capsule of Cryptococcus neoformans and the soluble form of its major polysaccharide, glucuronoxylomannan.
Incubation of encapsulated cryptococci with monoclonal antibodies (MAbs) specific for glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, produces two distinct capsular quellung-type reactions termed rim and puffy. The type of capsular reaction that occurs is determined by the epitope specificity of the MAb and the serotype of the yeast cell. Several biological activities, including opsonic activity, complement activation, and protective efficacy, are associated with the type of capsular reaction produced by a MAb. The goal of this study was to examine the reactivities of two families of anti-GXM MAbs with serotype A and D capsular polysaccharides in several immunochemical assays, including agglutination, immunofluorescence, quantitative precipitation, and enzyme-linked immunosorbent assay, in an effort to identify serological assays that are predictive of the capsular quellung reaction. The results showed that the type of capsular reaction (rim versus puffy) is a qualitative assessment of antibody-capsule interaction that cannot be predicted on the basis of a serological assay. The results further showed that antibody reactivity demonstrated in one serological assay is not necessarily predictive of results in another assay, particularly in cases where one assay examines antibody-capsule interactions, e.g., agglutination, and another assay examines interaction of antibody with soluble GXM. Taken together, the results suggest caution in interpretation of immunochemical assays for anti-GXM antibodies and recommend the use of multiple assays formats when studying anticryptococcal antibodies.
Animals↗
Monoclonal antibodies reactive with immunorecessive epitopes of glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans.
Cryptococcus neoformans is surrounded by an antiphagocytic capsule whose primary constituent is glucuronoxylomannan (GXM). An epitope shared by GXM serotypes A, B, C, and D is immunodominant when mice are immunized with serotype A GXM. In contrast, an epitope shared only by serotypes A and D is immunodominant when mice are immunized with serotype D. Hybridomas secreting antibodies reactive with subdominant epitopes were identified through a positive-negative screening procedure in which antibody-secreting colonies were characterized by reactivity with both the immunizing polysaccharide and GXMs from each of the four major serotypes. In this manner, a monoclonal antibody (MAb) that was reactive with an epitope shared only by serotypes A and B was identified and designated F10F5. Such an epitope has not been described previously. Immunization of mice with de-O-acetylated serotype A GXM generated a hybridoma that secreted an antibody, designated F12D2, that was reactive with all four serotypes. Unlike previously described monoclonal and polyclonal panspecific antibodies, the reactivity of MAb F12D2 was not altered by de-O-acetylation of GXM. These results indicate that there are at least two panspecific GXM epitopes; one epitope is dependent on O acetylation for antibody reactivity, and the other is independent of O acetylation. This study identifies strategies for production of MAbs that are reactive with subdominant or cryptic GXM epitopes and provides new information regarding the antigenic makeup and the humoral immune response to GXM, an essential virulence factor that is a target for active and passive immunization.
Animals↗
Cryptococcus neoformans-reactive and total immunoglobulin profiles of human immunodeficiency virus-infected and uninfected Ugandans.
We determined total and Cryptococcus neoformans glucuronoxylomannan (GXM)-reactive antibody repertoires of human immunodeficiency virus (HIV)-infected and HIV-uninfected Ugandans in a retrospective, case-control study of participants in a randomized controlled trial of pneumococcal vaccination. The study included 192 adults: 48 who subsequently developed cryptococcal meningitis (CM); (HIV+ CM+); 2 individuals who matched them in CD4+ T-cell level, stage of HIV disease, and age but did not develop CM (HIV+ CM-); and 48 HIV-uninfected individuals. Total serum immunoglobulin concentrations and titers of immunoglobulin M (IgM), IgG, and IgA to GXM, pneumococcal polysaccharides, and antibodies expressing certain V(H)3 idiotypes were determined with banked sera obtained before the development of cryptococcosis for HIV+ CM+ subjects. The results showed that HIV-infected subjects had significantly lower levels of IgM to GXM but higher levels of total immunoglobulin and IgG and IgA to GXM than those of HIV-uninfected subjects. HIV-infected subjects with a history of pneumonia had higher levels, and those with a history of herpes zoster had lower levels of GXM-binding antibodies than subjects with no history of either disease. Minimal to no cross-reactivity was demonstrated between antibodies to GXM and polysaccharides in a pneumococcal vaccine. No significant differences between the antibody repertoires of HIV+ CM+ and HIV+ CM- subjects were identified, but among subjects without a history of pneumonia, there was a trend towards lower V(H)3-positive antibody levels among HIV+ CM+ than among HIV+ CM- subjects. Our findings demonstrate an association between previous infectious diseases and differences in the total and GXM-reactive antibody repertoires of HIV-infected subjects and suggest the question of whether certain microbes modulate subsequent antibody responses to GXM deserves further study.
AIDS-Related Opportunistic Infections↗
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↗
Production of agglutinating monoclonal antibody against antigen 8 specific for Cryptococcus neoformans serotype D.
A hybridoma (clone CRND-8) that produced agglutinating monoclonal antibody (MAb) against Cryptococcus neoformans serotype D was established by using a soluble capsular polysaccharide-keyhole limpet hemocyanin conjugate for immunization. The isotype was immunoglobulin M(kappa). Specificity was determined by cell slide agglutination and enzyme-linked immunosorbent assay (ELISA). In both tests, the MAb reacted to serotypes D and A-D but not to serotypes A, B, and C. Furthermore, the specificity of the MAb determined by ELISA was the same as that of polyclonal antibody factor serum (PAb factor) 8, which showed high-level reactivity with serotypes D and A-D. These results supported the deduced specificity of the PAb-based antigenic factor 8. A total of 15 isolates of serotypes D and A-D but no serotype A isolates reacted with the MAb in cell slide agglutination tests. CRND-8 MAb can be used in place of PAb factor 8 for serotyping C. neoformans isolates and for the analysis of the antigen 8 epitope.
Agglutination Tests↗
Influence of different conditions on kinetics of tumor necrosis factor alpha release by peripheral blood mononuclear cells after stimulation with Cryptococcus neoformans: a possible explanation for different results.
In available literature, different kinetics for tumor necrosis factor alpha (TNF-alpha) release by peripheral blood mononuclear cells are reported upon stimulation with Cryptococcus neoformans. Results in this study showed that shaking cells gives faster kinetics of TNF-alpha release while large working volumes give lower TNF-alpha concentrations. Different experimental conditions thus influence kinetics of TNF-alpha release by phagocytes.
Cryptococcus neoformans↗
The different binding patterns of two immunoglobulin M monoclonal antibodies to Cryptococcus neoformans serotype A and D strains correlate with serotype classification and differences in functional assays.
Cryptococcus neoformans var. neoformans strains have historically been divided into serotypes A and D on the basis of reactivity with rabbit sera. Previously, we noted that two murine immunoglobulin M monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan produced different indirect immunofluorescence (IF) patterns, described as annular and punctate, when bound to C. neoformans cells from different strains. In this study, we examined the reactivity of these two MAbs, known as 12A1 and 13F1, with 20 C. neoformans var. neoformans strains, of which 13 were serotype A and 7 were serotype D. For all strains, MAb binding was studied by IF and agglutination assays. In addition, we blindly tested the IF patterns of 22 C. neoformans var. neoformans strains. For selected strains, MAb binding was studied by flow cytometry (FACScan) and phagocytosis assays. The epitopes recognized by MAbs 12A1 and 13F1 were found in all of the strains. MAb 12A1 binding produced an annular IF pattern with all of the strains, irrespective of the serotype classification. MAb 13F1 binding produced annular binding with all of the serotype A strains and punctate binding with 19 of 20 serotype D strains. In general, the punctate IF pattern was associated with lower fluorescence intensity, a requirement for higher antibody concentrations to produce yeast cell agglutination, and lower opsonic efficacy. Our results provide strong support for the existing classification of two serological types for strains assigned to variety neoformans and indicate qualitative and quantitative antigenic differences among serotype A and D strains.
Animals↗
Cryptococcus neoformans chemotyping by quantitative analysis of 1H nuclear magnetic resonance spectra of glucuronoxylomannans with a computer-simulated artificial neural network.
The complete assignment of the proton chemical shifts obtained by nuclear magnetic resonance (NMR) spectroscopy of de-O-acetylated glucuronoxylomannans (GXMs) from Cryptococcus neoformans permitted the high-resolution determination of the total structure of any GXM. Six structural motifs based on an alpha-(1-->3)-mannotriose substituted with variable quantities of 2-O-beta- and 4-O-beta-xylopyranosyl and 2-O-beta-glucopyranosyluronic acid were identified. The chemical shifts of only the anomeric protons of the mannosyl residues served as structure reporter groups (SRG) for the identification and quantitation of the six triads present in any GXM. The assigned protons for the mannosyl residues resonated at clearly distinguishable positions in the spectrum and supplied all the information essential for the assignment of the complete GXM structure. This technique for assigning structure is referred to as the SRG concept. The SRG concept was used to analyze the distribution of the six mannosyl triads of GXMs obtained from 106 isolates of C. neoformans. The six mannosyl triads occurred singularly or in combination with one or more of the other triads. The identification and quantitation of the SRG were simplified by using a computer-simulated artificial neural network (ANN) to automatically analyze the SRG region of the one-dimensional proton NMR spectra. The occurrence and relative distribution of the six mannosyl triads were used to chemotype C. neoformans on the basis of subtle variations in GXM structure determined by analysis of the SRG region of the proton NMR spectrum by the ANN. The data for the distribution of the six SRGs from GXMs of 106 isolates of C. neoformans yielded eight chemotypes, Chem1 through Chem8.
Bacterial Typing Techniques↗
Variants of a Cryptococcus neoformans strain elicit different inflammatory responses in mice.
The virulence of Cryptococcus neoformans isolates with high and low extracellular proteolytic activity was investigated in mice. No consistent relationship between proteolytic activity and virulence was observed, but isolates derived from one strain were shown to elicit different inflammatory responses.
Animals↗
Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans.
Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in approximately 10% of patients with AIDS. New selectable markers which confer resistance to G418 or phleomycin when transformed into C. neoformans were made. A hygromycin-selectable marker was modified to allow selection with a single copy of the marker.
Anti-Bacterial Agents↗
Cryptococcosis in the era of AIDS--100 years after the discovery of Cryptococcus neoformans.
Although Cryptococcus neoformans and cryptococcosis have existed for several millennia, a century has passed since the discovery of this encapsulated yeast and its devastating disease. With the advent of the AIDS pandemic, cryptococcal meningitis has emerged as a leading cause of infectious morbidity and mortality and a frequently life-threatening opportunistic mycosis among patients with AIDS. Both basic and clinical research have accelerated in the 1990s, and this review attempts to highlight some of these advances. The discussion covers recent findings, current concepts, controversies, and unresolved issues related to the ecology and genetics of C. neoformans; the surface structure of the yeast; and the mechanisms of host defense. Regarding cell-mediated immunity, CD4+ T cells are crucial for successful resistance, but CD8+ T cells may also participate significantly in the cytokine-mediated activation of anticryptococcal effector cells. In addition to cell-mediated immunity, monoclonal antibodies to the major capsular polysaccharide, the glucuronoxylomannan, offer some protection in murine models of cryptococcosis. Clinical concepts are presented that relate to the distinctive features of cryptococcosis in patients with AIDS and the diagnosis, treatment, and prevention of cryptococcosis in AIDS patients.
AIDS-Related Opportunistic Infections↗
Antibody response to Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan in patients after solid-organ transplantation.
Cryptococcosis is an important complication of solid-organ transplantation, but the risk factors for disease are poorly understood. The goal of this study was to investigate whether specific or nonspecific serum immunoglobulin levels determined in samples obtained before and after solid-organ transplantation differed in patients who did or did not develop cryptococcosis after transplantation. We analyzed pretransplantation sera from 25 subjects, 15 who subsequently developed cryptococcosis and 10 who did not, and posttransplantation sera from 24 subjects, 13 who developed cryptococcosis and 11 who did not. All subjects received a tacrolimus-based immunosuppressive regimen. Total immunoglobulin levels were measured by immunodiffusion, and Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM)-specific serum antibody levels were determined by enzyme-linked immunosorbent assays. The results showed that solid-organ transplantation had a significant effect on total immunoglobulin and GXM-reactive antibody levels. GXM-reactive antibody levels differed in subjects who did and did not develop cryptococcosis. In pretransplant serum samples, the levels of GXM-reactive immunoglobulin M (IgM) were significantly lower in subjects who developed cryptococcosis after transplantation than in those who did not. For posttransplant serum samples, the levels of GXM-reactive IgM and IgG were significantly higher among the subjects who developed cryptococcosis than among those who did not. These findings suggest that perturbations in the preexisting antibody or B-cell repertoire and/or related to treatment of rejection, transplantation, or immunosuppressive therapy could translate into an increased risk for transplant-associated cryptococcosis.
Adult↗
Transcription factor STE12alpha has distinct roles in morphogenesis, virulence, and ecological fitness of the primary pathogenic yeast Cryptococcus gattii.
Cryptococcus gattii is a primary pathogenic yeast, increasingly important in public health, but factors responsible for its host predilection and geographical distribution remain largely unknown. We have characterized C. gattii STE12alpha to probe its role in biology and pathogenesis because this transcription factor has been linked to virulence in many human and plant pathogenic fungi. A full-length STE12alpha gene was cloned by colony hybridization and sequenced using primer walk and 3' rapid amplification of cDNA ends strategies, and a ste12alpha delta gene knockout mutant was created by URA5 insertion at the homologous site. A semiquantitative analysis revealed delayed and poor mating in ste12alpha delta mutant; this defect was not reversed by exogenous cyclic AMP. C. gattii parent and mutant strains showed robust haploid fruiting. Among putative virulence factors tested, the laccase transcript and enzymatic activity were down regulated in the ste12alpha delta mutant, with diminished production of melanin. However, capsule, superoxide dismutase, phospholipase, and urease were unaffected. Similarly, Ste12 deficiency did not cause any auxotrophy, assimilation defects, or sensitivity to a large panel of chemicals and antifungals. The ste12alpha delta mutant was markedly attenuated in virulence in both BALB/c and A/Jcr mice models of meningoencephalitis, and it also exhibited significant in vivo growth reduction and was highly susceptible to in vitro killing by human neutrophils (polymorphonuclear leukocytes). In tests designed to simulate the C. gattii natural habitat, the ste12alpha delta mutant was poorly pigmented on wood agar prepared from two tree species and showed poor survival and multiplication in wood blocks. Thus, STE12alpha plays distinct roles in C. gattii morphogenesis, virulence, and ecological fitness.
Animals↗
The Cryptococcus neoformans catalase gene family and its role in antioxidant defense.
In the present study, we sought to elucidate the contribution of the Cryptococcus neoformans catalase gene family to antioxidant defense. We employed bioinformatics techniques to identify four members of the C. neoformans catalase gene family and created mutants lacking single or multiple catalase genes. Based on a phylogenetic analysis, CAT1 and CAT3 encode putative spore-specific catalases, CAT2 encodes a putative peroxisomal catalase, and CAT4 encodes a putative cytosolic catalase. Only Cat1 exhibited detectable biochemical activity in vitro, and Cat1 activity was constitutive in the yeast form of this organism. Although they were predicted to be important in spores, neither CAT1 nor CAT3 was essential for mating or spore viability. Consistent with previous studies of Saccharomyces cerevisiae, the single (cat1, cat2, cat3, and cat4) and quadruple (cat1 cat2 cat3 cat4) catalase mutant strains exhibited no oxidative-stress phenotypes under conditions in which either exogenous or endogenous levels of reactive oxygen species were elevated. In addition, there were no significant differences in the mean times to mortality between groups of mice infected with C. neoformans catalase mutant strains (the cat1 and cat1 cat2 cat3 cat4 mutants) and those infected with wild-type strain H99. We conclude from the results of this study that C. neoformans possesses a robust antioxidant system, composed of functionally overlapping and compensatory components that provide protection against endogenous and exogenous oxidative stresses.
Animals↗
Transcription factor Nrg1 mediates capsule formation, stress response, and pathogenesis in Cryptococcus neoformans.
The Cryptococcus neoformans NRG1 gene was identified using gene microarrays to define putative transcription factor genes regulated by the cyclic AMP (cAMP) signal transduction pathway. Disruption of NRG1 results in delayed capsule formation and mating, two phenotypes that are directly controlled by cAMP signaling. Putative targets of the Nrg1 transcription factor were identified using a second genome microarray to define differences in the transcriptomes of the wild-type and nrg1 mutant strains. These experiments implicate Nrg1 in the transcriptional control of multiple genes involved in carbohydrate metabolism and substrate oxidation, as well as the UGD1 gene encoding a UDP-glucose dehydrogenase required for polysaccharide capsule production and cell wall integrity. In addition to being under transcriptional control of the cAMP pathway, Nrg1 contains a putative protein kinase A phosphorylation site; mutation of this motif results in reduced Nrg1 activity. Consistent with prior studies in hypocapsular mutants, the nrg1 mutant strain is attenuated in an animal model of disseminated cryptococcal disease.
Amino Acid Sequence↗