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PubMed1992

Galactoxylomannans of Cryptococcus neoformans.

Galactoxylomannans (GalXMs) from single isolates of Cryptococcus neoformans serotypes A, B, and D were isolated from culture supernatants and then purified by affinity, ion-exchange, and gel-filtration chromatography. GalXMs are a group of closely related complex polysaccharides. GalXMs from serotypes A (9759 A) and C (3183 C) and an acapsular mutant of serotype D (Cap67 D) have similar galactose, xylose, and mannose molar ratios, but each has some unique structural features. GalXM9759 A and GalXM 3183 C were associated with a starchlike glucan that was removed during purification. Only a trace of glucose was detected in the Cap67 D GalXM. Gas-liquid chromatography-mass spectroscopy of per-O-methylated polysaccharides and 13C nuclear magnetic resonance spectroscopy showed that GalXM is a complex branched polysaccharide. The main chain consists of mannose or galactose or alternating mannose and galactose residues. Xylose is present only as nonreducing termini. Galactofuranose occurs only in 3183 C and Cap67 D, and it is always present as nonreducing termini.

Carbohydrates↗
PubMed1992

Molecular and genetic analysis of URA5 transformants of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans ura5 mutants were transformed with linearized or circular plasmids containing the C. neoformans orotidine monophosphate pyrophosphorylase gene. Following electroporation, randomly isolated transformants were analyzed for the mitotic and meiotic stability of uracil prototrophy. All stable transformants tested showed nonspecific ectopic integration. Uracil prototrophy in these transformants was stable through meiosis. Some of the stable transformants showed integration of both URA5 and vector sequences, while others lacked any vector sequences. Unstable transformants exhibited the presence of an autonomously replicating plasmid which had undergone significant sequence rearrangement. The autonomously replicating plasmid in the transformants was observed to be the same size or smaller than the transforming plasmid, was maintained in a linear form, and had acquired a genomic sequence(s) with homology to a sequence(s) on all the chromosomes. The conservation of a 300-bp sequence at the 5' end of the URA5 gene was observed in all the rearranged plasmids. These results suggest mechanisms of plasmid maintenance in C. neoformans that are different from those reported for other yeasts. The ura5 mutant was significantly less virulent than the wild type. The transformants did not recover virulence regardless of prototrophic stability.

Chromosome Mapping↗
PubMed1992

Heterogeneity of phenol oxidases in Cryptococcus neoformans.

Phenol oxidase enzymes, linked to virulence in Cryptococcus neoformans, were prepared from broken cells. More enzyme activity was found in the ultracentrifugation supernatant; less was found in the membrane fraction. Phenol oxidases were located in acrylamide gel electropherograms by activity staining with L-dihydroxyphenylalanine (DOPA). Mobility differences between soluble and solubilized membrane-bound phenol oxidases were not found. Comparison of enzymes produced at 25 and 37 degrees C revealed that the enzyme had lower activity and lower mobility at 37 degrees C. The mobility of 25 degrees C phenol oxidases from strains of C. neoformans var. gattii was lower than that of those from C. neoformans var. neoformans. Half of the phenol oxidase produced at 25 degrees C was bound by concanavalin A, while that produced at 37 degrees C was not bound. However, glucose starvation of cultures at 25 degrees C overnight resulted in increased amounts of enzyme which did not bind to concanavalin A. A given strain of C. neoformans produces different species of phenol oxidase under different culture conditions.

Cryptococcus neoformans↗
PubMed1992

In vivo complement activation and binding of C3 to encapsulated Cryptococcus neoformans.

Tissues from mice infected with Cryptococcus neoformans were examined by immunofluorescence to determine the extent of deposition of complement component C3 on encapsulated cryptococci. The relative percentages of cryptococci in each tissue having readily visible C3 were greatest for liver and lung tissues, with the kidney tissue having the next highest percentage and the spleen having the lowest percentage. Binding of C3 fragments to cryptococci in brain tissue was essentially absent.

Animals↗
PubMed1993

Accelerated decay of C3b to iC3b when C3b is bound to the Cryptococcus neoformans capsule.

Incubation of encapsulated and nonencapsulated Cryptococcus neoformans in normal human serum (NHS) leads to activation and binding of potentially opsonic fragments of complement component C3 to the yeast cells. Analysis of the molecular forms of C3 after incubation of encapsulated cryptococci in NHS showed that the percentage of bound C3 occurring as iC3b approached 100% after 8 min. The percentage of bound C3 occurring as iC3b on nonencapsulated cryptococci never exceeded 70%, even after 60 min of incubation in NHS. Conversion of C3b to iC3b was assessed further by incubating C3b-coated cryptococci for various times with a mixture of complement factors H and I at 40% of their respective physiological concentrations. Most, if not all, of the C3b on encapsulated cryptococci was converted to iC3b at a single fast rate. Conversion of C3b to iC3b on nonencapsulated cryptococci did not follow a single rate constant and appeared to have a fast and a slow component. Studies of the requirements for factors H and I in cleavage of C3b to iC3b showed steep dose-response curves for both factors in the case of encapsulated cryptococci and shallow curves with C3b bound to nonencapsulated cryptococci. Taken together, our results indicate that C3b molecules bound to encapsulated cryptococci have a uniformly high susceptibility to conversion to iC3b by factors H and I. In contrast, a significant portion of the C3b bound to nonencapsulated cryptococci is very resistant to conversion to iC3b by factors H and I.

Complement C3b↗
PubMed1993

The gene encoding phosphoribosylaminoimidazole carboxylase (ADE2) is essential for growth of Cryptococcus neoformans in cerebrospinal fluid.

A cryptococcal meningitis model in corticosteroid-treated rabbits was used to assess the requirement for the phosphoribosylaminoimidazole gene (ADE2) for virulence of Cryptococcus neoformans. A wild-type strain (H99), an ade2 auxotroph of H99 (M001), and a randomly selected prototrophic transformant of M001 (M001.1c) which had received the cloned ADE2 cDNA copy were inoculated intrathecally into immunosuppressed rabbits. While M001 was avirulent in the central nervous system model, virulence was completely restored to wild-type pathogenicity in the prototrophic transformant. This study identifies the pathogenic importance of an endogenous adenine pathway in this yeast and confirms that purine biosynthesis is a potential target for antifungal therapy. It also demonstrates that the virulence of C. neoformans can be molecularly changed and detected within a clinically relevant animal model.

Animals↗
PubMed1993

Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen.

The mobility of human neutrophils (PMN) in response to encapsulated or nonencapsulated Cryptococcus neoformans cells or cryptococcal culture filtrate (CneF) and its components was studied by using a 48-well modified Boyden chamber. Encapsulated C. neoformans (isolate 184A) cells and CneF-184A stimulated directed migration of human PMN in the absence of serum (direct chemotactic activity) and activated a heat-labile component(s) in fresh human serum to become a chemoattractant(s) for human PMN (indirect chemotactic activity). At a 1:8 dilution (0.25 mg of carbohydrate per ml), CneF-184A displayed chemokinetic activity when assessed with a checkerboard assay. Nonencapsulated C. neoformans isolate 602 cells did not have direct chemotactic activity but did have indirect chemotactic activity. The capsule of C. neoformans is composed predominantly of glucuronoxylomannan (GXM). Purified GXM displayed both direct and indirect chemotactic activity. CneF-184A contains, in addition to GXM, a concanavalin A-binding mannoprotein (MP), whereas CneF-602 contains no GXM but does contain MP. CneF-184A showed direct chemotactic activity and CneF-602 did not. Both CneF-184A and CneF-602 displayed indirect chemotactic activity for human PMN. In addition, purified MP from CneF-184A, like CneF-602, showed only indirect chemotactic activity. These results indicate that GXM contributes to the direct chemotactic activity of PMN observed with the whole encapsulated yeast cells and the unfractionated CneF derived from the encapsulated cells. Both MP and GXM from encapsulated C. neoformans cells mediate indirect chemotactic activity on human PMN.

Antigens, Fungal↗
PubMed1993

Effect of polysaccharide capsule and methods of preparation on human lymphocyte proliferation in response to Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic encapsulated yeast that infects patients that have defective cell-mediated immunity, including AIDS patients. Whole cryptococcal organisms that are killed by heating stimulate normal human lymphocytes to proliferate. However, strains of C. neoformans vary widely in virulence and therefore in their ability to cause disease in humans. To determine the effect of virulence factors such as the cryptococcal capsule, serotype, and the state of the organisms on the lymphocyte response to C. neoformans, human peripheral blood mononuclear cells were stimulated with C. neoformans in vitro and lymphocyte proliferation was determined. The major determinant of the lymphocyte response to C. neoformans was the amount of polysaccharide present. The response was greater after stimulation by minimally encapsulated strains (strains C3D, 68, and 613) than by heavily encapsulated strains (strains 6 and 145). A heavily encapsulated strain (strain 6) did not suppress the response to an acapsular mutant (strain 67). However, the response to an acapsular strain was suppressed by the addition of purified polysaccharide. Human lymphocytes responded to both serotypes of C. neoformans var. neoformans. The antigen responsible for lymphocyte stimulation was preserved despite various techniques of inactivation, including heat, paraformaldehyde fixation, irradiation, and mechanical disruption. Finally, lymphocytes responded equally to live and killed organisms. These results suggest that capsular polysaccharide, a known virulence factor, may suppress the human lymphocyte response to C. neoformans during an infection. Lymphocytes could respond to C. neoformans regardless of the viability of the organism, and they could also respond to disrupted organisms. We speculate that lymphocyte proliferation in vitro could be related to the protective immune response in host defense to C. neoformans and that it is suppressed by virulence factors of C. neoformans.

Cells, Cultured↗
PubMed1993

T-cell-dependent and T-cell-independent mechanisms of tolerance to glucuronoxylomannan of Cryptococcus neoformans serotype A.

Glucuronoxylomannan (GXM), a type 2 T-independent antigen, is the major component of the capsular polysaccharide (CnCAP) of Cryptococcus neoformans. Previous studies have described the tolerogenic effects of high doses of CnCAP on the specific humoral response. In this investigation, evidence for both high-dose and low-dose tolerance to GXM is presented. BALB/cBy female mice, primed with either 5 ng or 50 micrograms of GXM, then coimmunized 3 days later with immunogenic doses of both GXM and type 3 pneumococcal polysaccharide (SSS-III), showed an antigen-specific inhibition in their splenic plaque-forming cell (PFC) responses to GXM compared with control groups primed with normal saline. SSS-III PFCs remained unchanged between GXM-primed and normal saline-primed groups. Low-dose tolerance appeared to be T dependent, whereas high-dose tolerance appeared to be T independent. Low-dose tolerance to GXM could not be induced in athymic BALB/c nu/nu mice, whereas high-dose tolerance in the same mice could be induced. Furthermore, low-dose tolerance was adoptively transferred with B-cell-depleted splenocytes to naive BALB/c mice, while high-dose tolerance was not. Complement-mediated depletion of CD4+ but not CD8+ splenocytes from low-dose-primed mice abrogated the transfer of low-dose tolerance. These findings indicate T-dependent and T-independent mechanisms of antigen-specific B-cell tolerance to GXM in BALB/c mice at low and high antigen doses, respectively.

Animals↗
PubMed1993

Specificity of Cryptococcus neoformans factor sera determined by enzyme-linked immunosorbent assay and dot enzyme assay.

An indirect enzyme-linked immunosorbent assay (ELISA) and a dot enzyme assay (DEA) were used to determine the specificities of Cryptococcus neoformans factor sera to serotype type-specific capsular polysaccharides, glucuronoxylomannans (GXMs). Pure and chemically characterized GXMs were obtained from representative isolates of C. neoformans serotypes A, B, C, and D. Distinctive specificity patterns and quantitative differences were observed for each factor serum when the selected GXMs were studied by ELISA. The specificity patterns for each factor serum determined by DEA almost completely paralleled the ELISA results. The serotype specificities demonstrated by ELISA and DEA were similar to previously reported results that were obtained by slide agglutination studies of whole cells. On the basis of the ELISA and DEA activity patterns, factor sera 5, 6, and 8 were specific for serotypes B, C, and D, respectively; factor serum 1 was strongly reactive to all serotypes; factor serum 2 was specific for serotypes A, B, and D; factor serum 3 was specific for serotypes A and D; and factor serum 4 was specific for serotypes B and C. The specificity of factor serum 7 for serotype A was demonstrated by DEA only. Structural variation was indicated among the serotype C isolates studied because a unique activity pattern versus factor serum 6 was observed for each isolate. The quantitative differences in the activity of the GXMs from five serotype C isolates suggest that mannopyranoside residues substituted O-2 and O-4 with xylose are essential elements of the determinant responsible for the observed activity of factor 6. No significant differences in activity patterns and specificities of factor serum 6 were observed when O-deacetylated GXMs were substituted for the native GXMs. Our results show that ELISA and DEA are valuable techniques for the serological analysis of cryptococcal factor sera and GXMs.

Animals↗
PubMed1993

Effects of strain variation, serotype, and structural modification on kinetics for activation and binding of C3 to Cryptococcus neoformans.

Incubation of encapsulated cells of Cryptococcus neoformans in normal human serum leads to activation of the alternative complement pathway and deposition of opsonic fragments of C3 into the capsule. We determined whether the variation in capsular structure that occurs among the four major cryptococcal serotypes was reflected in the kinetics for activation and binding of C3. We also examined the effects on activation kinetics of de-O-acetylation or periodate oxidation of the capsule. Binding kinetics were characterized in terms of the time required to deposit 5% of the maximal amount of C3 on the yeast (t5%), the first-order rate constant for amplification of C3 deposition (k'), and the maximum amount of C3 that could be deposited in the capsule (C3max). Our results showed that variations in the capsular structure that characterized each serotype had no significant influence on C3max but that the rate of C3 deposition depended significantly on the serotype. C3 accumulated at a higher rate on cells of serotypes A and D than on cells of serotypes B and C. There was a significant correlation between capsular volume and C3max, although the relationship was not linear. Periodate treatment of encapsulated cryptococci of all four serotypes led to decapsulation. Periodate-oxidized encapsulated cells displayed kinetics for activation and binding of C3 that were identical to kinetics observed with nonencapsulated cryptococci. Finally, de-O-acetylation led to a significant but relatively minor increase in C3max.

Acetylation↗
PubMed1994

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans.

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.

Antigens, Fungal↗
PubMed1994

Occurrences, specificities, and functions of ubiquitous antibodies in human serum that are reactive with the Cryptococcus neoformans cell wall.

Previous studies found that normal human serum (NHS) contains an immunoglobulin G (IgG) antibody that mediates initiation of the classical complement pathway by nonencapsulated Cryptococcus neoformans. The present study used an enzyme-linked immunosorbent assay with whole nonencapsulated yeast cells as solid-phase antigens to demonstrate the presence of high levels of IgG antibody in each of 11 sera from normal adult donors. The IgG antibodies were of the IgG2 subclass. The antibody activity was blocked completely by treatment of serum with isolated yeast glucan. Treatment of serum with mannan or chitin had no effect on antibody levels. Antibody activity was adsorbed completely by treatment of serum with zymosan particles. Adsorption with intact cells of Saccharomyces cerevisiae or Candida albicans had no effect, suggesting that the glucan on S. cerevisiae or C. albicans is not surface exposed. Assessment of the opsonic requirements for phagocytosis of nonencapsulated cryptococci by monocyte-derived human macrophages (MO-M phi) showed that high levels of phagocytosis occurred when yeast cells were opsonized with NHS. Removal of anti-glucan antibody by adsorption with whole nonencapsulated cryptococci did not diminish opsonic activity. Heat-inactivated serum or anti-glucan antibody affinity purified from NHS lacked opsonic activity. Taken together, these results indicate that phagocytosis of nonencapsulated cryptococci by monocyte-derived human macrophages has an obligatory requirement for opsonic ligands of the complement system, with no contribution by the anti-glucan IgG that is found in NHS.

Antibodies, Fungal↗
PubMed1994

Pathogenesis of pulmonary Cryptococcus neoformans infection in the rat.

The pathogenesis of Cryptococcus neoformans pulmonary infection in the rat was studied after intratracheal inoculation. Lungs were examined at various times following infection for histopathology in conjunction with macrophage markers, proliferating cell nuclear antigen (PCNA), and capsular glucuronoxylomannan (GXM) antigen. Serum GXM, immunoglobulin M (IgM) and IgG titers and organ fungal burden were compared with pathological findings. C. neoformans organisms were in the lung parenchyma 2 h postinoculation, and GXM antigen was present in surrounding tissues shortly thereafter. Extrapulmonary dissemination occurred early in infection. Two phases of host cellular inflammatory response were discernible: early local macrophage recruitment at 2 to 4 days followed by granulomatous inflammation, which reached maximum intensity 14 days after infection. The granulomatous phase was preceded by lymphocyte influx with macrophage proliferation and maturation into epithelioid histiocytes; this was paralleled by a shift of yeasts from extracellular to intracellular spaces. Tissue IgG deposits, serum IgG to GXM, and localization of tissue GXM immunoreactivity to epithelioid cells were noted at 2 to 4 weeks. A 10-fold decrease in lung fungal burden occurred 25 days postinfection and was associated with resolving granulomas, fewer proliferating cells, and decreased tissue GXM. The present study demonstrates that (i) C. neoformans penetrates the lung parenchyma shortly after infection; (ii) immunocompetent rats control pulmonary cryptococcosis efficiently, with minimal extrapulmonary dissemination and low levels of serum GXM; and (iii) macrophage activation is likely to play a crucial role in limiting C. neoformans infection in the rat lung.

Animals↗
PubMed1994

Genetic study of oxygen resistance and melanization in Cryptococcus neoformans.

Genetic analysis of oxygen-sensitive mutants of Cryptococcus neoformans revealed two loci (oxy1 and oxy2) linking hyperoxia sensitivity to production of melanin, a known virulence factor. Hyperoxia-sensitive strain 562 (oxy1 oxy2) is albino and avirulent. oxy2-defective strains lacking the oxy1 defect are melanin deficient but show normal hyperoxia resistance. Mutants defective at three additional mapped melanin loci fail to show hyperoxia sensitivity in the oxy1 background. Revertants of strain 562, which regain the ability to synthesize melanin by mutation at suppressor sites unlinked to oxy2, retain the oxygen sensitivity conferred by their oxy1 and oxy2 defects. These data identify the melanin gene oxy2 as unique in its association of hyperoxia resistance and melanization.

Chromosome Mapping↗
PubMed1994

Monoclonal antibodies to Cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice.

Immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan (GXM) were studied for their ability to modify the course of intravenous Cryptococcus neoformans infection in mice. A/J mice were given intraperitoneal injection of 1.0 mg of either a GXM-binding IgG1 MAb (2H1 or 2D10 gamma 1) or the irrelevant isotype-matched control MAb 36-65 prior to intravenous infection. Parameters used to study antibody efficacy were lung and brain tissue fungal burden, lung and brain weights, serum GXM levels, and histopathological examination of lung, brain, heart, kidney, and spleen tissues. Mice given GXM-binding MAb had significantly reduced lung tissue fungal burden as measured by CFU. In contrast to the reduction in lung tissue burden, the reduction in brain tissue burden was small and did not achieve statistical significance. Serum GXM levels were reduced in mice receiving GXM-binding MAb. Histopathological examination revealed reduced numbers of granulomas and C. neoformans organisms in the lungs, brains, and kidneys of MAb 2H1-treated mice relative to control mice. The lungs and brains of mice receiving GXM-binding MAb weighed significantly less than those of control animals, consistent with the reduced inflammation noted histologically. Subendocardial inflammation and kidney cortical infarctions were present in control infected mice but not in MAb 2H1-treated mice. Immunocytochemical staining for polysaccharide antigen revealed a marked reduction in the amount of tissue polysaccharide in mice treated with MAb 2H1 relative to control mice. The results support an useful role for passive antibody administration in C. neoformans infections.

Animals↗
PubMed1994

Production of tumor necrosis factor alpha in human leukocytes stimulated by Cryptococcus neoformans.

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of inflammation and may promote human immunodeficiency virus replication in latently infected cells. Since cryptococcosis often is associated with aberrations in the host inflammatory response and occurs preferentially in persons with AIDS, we defined the conditions under which human leukocytes produce TNF-alpha when stimulated by Cryptococcus neoformans. Peripheral blood mononuclear cells (PBMC) produced comparable amounts of TNF-alpha following stimulation with C. neoformans and lipopolysaccharide. Detectable TNF-alpha release in response to C. neoformans occurred only when fungi with small-sized capsules were used and complement-sufficient serum was added. Fractionation of PBMC established that monocytes were the predominant source of TNF-alpha. TNF-alpha gene expression and release occurred significantly later in PBMC stimulated with C. neoformans than in PBMC stimulated with LPS. C. neoformans was also a potent inducer of TNF-alpha from freshly isolated bronchoalveolar macrophages (BAM). Upon in vitro culture, BAM and monocytes bound greater numbers of fungal cells, yet their capacity to produce TNF-alpha following cryptococcal stimulation declined by 74 to 100%. However, this decline was reversed if the BAM and monocytes were cultured with gamma interferon. These data establish that C. neoformans can potently stimulate TNF-alpha release from human leukocytes. However, several variables profoundly affected the amount of TNF-alpha released, including the type of leukocyte and its state of activation, the size of the cryptococcal capsule, and the availability of opsonins.

Cryptococcus neoformans↗