Capsular localization of the Cryptococcus neoformans polysaccharide component galactoxylomannan

Cryptococcus neoformans capsular polysaccharide is composed of at least two components, glucuronoxylomannan (GXM) and galactoxylomannans (GalXM). Although GXM has been extensively studied, little is known about the location of GalXM in the C. neoformans capsule, in part because there are no serological reagents specific to this antigen. To circumvent the poor immunogenicity of GalXM, this antigen was conjugated to protective antigen from Bacillus anthracis as a protein carrier. The resulting conjugate elicited antibodies that reacted with GalXM in mice and yielded an immune serum that proved useful for studying GalXM in the polysaccharide capsule. In acapsular cells, immune serum localized GalXM to the cell wall. In capsulated cells, immune serum localized GalXM to discrete pockets near the capsule edge. GalXM was abundant on the nascent capsules of budding daughter cells. The constituent sugars of GalXM were found in vesicle fractions consistent with vesicular transport for this polysaccharide. In addition, we generated a single-chain fraction variable fragment antibody with specificity to oxidized carbohydrates that also produced punctate immunofluorescence on encapsulated cells that partially colocalized with GalXM. The results are interpreted to mean that GalXM is a transient component of the polysaccharide capsule of mature cells during the process of secretion. Hence, the function of GalXM appears to be more consistent with that of an exopolysaccharide than a structural component of the cryptococcal capsule.     

Equatorial ring-like channels in the Cryptococcus neoformans polysaccharide capsule

Under certain conditions, India ink particles can penetrate the capsule of the opportunistic pathogen Cryptococcus neoformans. India ink penetration gave two distinct patterns, one as a ring in the middle of the capsule, and another as a double spot located at opposite poles of the cells. These spots were perpendicularly orientated to the bud. This pattern suggests the existence of a localized structure deep in the capsule that can accumulate large insoluble particles. Although the mechanisms responsible for the assemblage and maintenance of ring-like channels are not understood, their existence deep within the capsule implies a new level of complexity for this enigmatic structure.     

Induction by Klebsiella aerogenes of a Melanin-Like Pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37°C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.     

Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.     

The volume and hydration of the Cryptococcus neoformans polysaccharide capsule

We present a new method to measure capsule size in the human fungal pathogen Cryptococcus neoformans that avoids the limitations and biases inherent in India ink measurements. The method is based on the use of gamma-radiation, which efficiently releases the capsule from the cell. By comparing the volume of irradiated and non-irradiated cells, one can accurately estimate the relative size of the capsule per cell. This method was also used to obtain an estimate of the capsule weight and water content. The C. neoformans capsule is a highly hydrated structure in all the conditions measured. However, after capsule enlargement, the amount of capsular polysaccharide significantly increases, suggesting a that capsule growth has a high energy cost for the cell.     

Chemical characterization of capsular polysaccharide from Cryptococcus neoformans serotype A-D

During a study of serotyping of Cryptococcus neoformans, we found that the type strain of C. neoformans (CBS 132) was serotype A-D. This strain agglutinated with both factor 7 serum (specific for serotype A) and factor 8 serum (specific for serotype D) in our serotyping system. Therefore, we investigated the chemical structure of the antigenic capsular polysaccharide of this strain. The soluble capsular polysaccharide was obtained from the culture supernatant fluid by precipitation with ethanol. Column chromatography of the polysaccharide on DEAE-cellulose yielded three fractions (F-1 to F-3). The major antigenic activity was found in the F-3 fraction. The results obtained by methylation analysis, controlled Smith degradation-methylation analysis, partial acid hydrolysis, and other structural studies of F-3 polysaccharide indicated that the polysaccharide contains mannose, xylose, and glucuronic acid at a ratio of 7:2:2, and has a backbone of alpha (1-3)-linked D-mannopyranoside residues with a single branch of beta (1-2)-xylose and glucuronic acid. The ratio of mannose residues with or without a branch in the F-3 polysaccharide was 4:3 and its molecular weight calculated from the average of the degree of polymerization was 46,500 daltons. These results indicate that the chemical structure of the capsular polysaccharide of serotype A-D is very similar to those from serotypes A and D, suggesting that small differences in the molar ratio and pattern of linkage of monosaccharides in the branch of the polysaccharides of the three serotypes may be responsible for their different specificities.     

UDP glucuronate decarboxylase and synthesis of capsular polysaccharide in Cryptococcus neoformans

UDP glucuronate decarboxylase activity was comparable in encapsulated and non-encapsulated strains of Cryptococcus neoformans, required NAD (K_a = 0.2 mM), and was inhibited by NADH (K_i = 0.1 mM) and UDP xylose.     

Toll-like receptor 4 mediates intracellular signaling without TNF-alpha release in response to Cryptococcus neoformans polysaccharide capsule

Toll-like receptors (TLR) 2 and 4 are cell surface receptors that in association with CD14 enable phagocytic inflammatory responses to a variety of microbial products. Activation via these receptors triggers signaling cascades, resulting in nuclear translocation of NF-kappa B and a proinflammatory response including TNF-alpha production. We investigated whether TLRs participate in the host response to Cryptococcus neoformans glucuronoxylomannan (GXM), the major capsular polysaccharide of this fungus. Chinese hamster ovary fibroblasts transfected with human TLR2, TLR4, and/or CD14 bound fluorescently labeled GXM. The transfected Chinese hamster ovary cells were challenged with GXM, and activation of an NF-kappa B-dependent reporter construct was evaluated. Activation was observed in cells transfected with both CD14 and TLR4. GXM also stimulated nuclear NF-kappa B translocation in PBMC and RAW 264.7 cells. However, stimulation of these cells with GXM resulted in neither TNF-alpha secretion nor activation of the extracellular signal-regulated kinase 1/2, p38, and stress-activated protein kinase/c-Jun N-terminal kinase mitogen-activated protein kinase pathways. These findings suggest that TLRs, in conjunction with CD14, function as pattern recognition receptors for GXM. Furthermore, whereas GXM stimulates cells to translocate NF-kappa B to the nucleus, it does not induce activation of mitogen-activated protein kinase pathways or release of TNF-alpha. Taken together, these observations suggest a novel scenario whereby GXM stimulates cells via CD14 and TLR4, resulting in an incomplete activation of pathways necessary for TNF-alpha production.     

Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.     

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.     

Intracellular parasitism of macrophages by Cryptococcus neoformans

Cryptococcus neoformans, an encapsulated fungal pathogen, causes meningoencephalitis in immunocompromised patients. Recent in vivo studies have demonstrated that C. neoformans is a facultative intracellular pathogen, as was previously suggested by in vitro studies. For survival in macrophages, C. neoformans utilizes a novel strategy for intracellular parasitism that includes the accumulation of intracellular polysaccharide in cytoplasmic vesicles. Confirmation of the fact that C. neoformans is a facultative intracellular pathogen could provide new insights into several poorly understood areas of cryptococcal pathogenesis, including mechanisms for latency and persistence and the lack of efficacy of humoral immunity. The finding that C. neoformans replicates inside macrophages in vitro in a manner similar to that observed in vivo provides an excellent system to dissect the molecular mechanisms responsible for this unique pathogenic strategy.     

Micromorphology of Cryptococcus neoformans

Fine details of the internal and external morphology of Cryptococcus neoformans as seen in ultrathin sections are described and illustrated with electron micrographs. The capsule characteristic of this species contained microfibrils (30 to 40 A in diameter) that appeared to radiate from the cell wall and to coil and intertwine in various directions. These thin, uniformly structured, electron-dense filaments are believed to represent complex polysaccharide molecules. The internal morphology of C. neoformans was in many ways similar to that of yeasts studied by other authors. The cell was uninucleate with a single nucleolus. The nuclear envelope, a pair of unit membranes interrupted by pores, was typical of that found in eucaryotic organisms. Smooth endoplasmic reticulum, mitochondria, vacuoles, storage granules, and ribosomes were consistent features of the cytoplasm. In addition, C. neoformans presented membranous organelles derived from the plasma membrane and comparable to bacterial mesosomes and mitochondria of an annulate type.     

Epidemiology of Cryptococcus neoformans

The concept of the epidemiology of Cryptococcus neoformans as the causative agent of cryptococcosis and as a basidiomycetous yeast is based on the fact that bird manure has been until now its only known habitat but not plant material which likewise harbours various non-pathogenic Cryptococcus species.It could be shown that the possible influence of nutritional factors on the morphology and morphogenesis earns attention not only in view of the epidemiology of C. neoformans but of its perfect states, too.     

Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised; the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25°C and 37°C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.     

Cryptococcus neoformans carried by Odontomachus bauri ants

Cryptococcus neoformans is the most common causative agent of cryptococcosis worldwide. Although this fungus has been isolated from a variety of organic substrates, several studies suggest that hollow trees constitute an important natural niche for C. neoformans. A previously surveyed hollow of a living pink shower tree (Cassia grandis) positive for C. neoformans in the city of Rio de Janeiro, Brazil, was chosen for further investigation. Odontomachus bauri ants (trap-jaw ants) found inside the hollow were collected for evaluation as possible carriers of Cryptococcus spp. Two out of 10 ants were found to carry phenoloxidase-positive colonies identified as C. neoformans molecular types VNI and VNII. The ants may have acted as a mechanical vector of C. neoformans and possibly contributed to the dispersal of the fungi from one substrate to another. To the best of our knowledge, this is the first report on the association of C. neoformans with ants of the genus Odontomachus.