Cryptococcal xylosyltransferase 1 (Cxt1p) from Cryptococcus neoformans plays a direct role in the synthesis of capsule polysaccharides

The opportunistic yeast Cryptococcus neoformans causes serious disease in humans and expresses a prominent polysaccharide capsule that is required for its virulence. Little is known about how this capsule is synthesized. We previously identified a beta1,2-xylosyltransferase (Cxt1p) with in vitro enzymatic activity appropriate for involvement in capsule synthesis. Here, we investigate C. neoformans strains in which the corresponding gene has been deleted (cxt1Delta). Loss of CXT1 does not affect in vitro growth of the mutant cells or the general morphology of their capsules. However, NMR structural analysis of the two main capsule polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), showed that both were missing beta1,2-xylose residues. There was an approximately 30% reduction in the abundance of this residue in GXM in mutant compared with wild-type strains, and mutant GalXM was almost completely devoid of beta1,2-linked xylose. The GalXM from the mutant strain was also missing a beta1,3-linked xylose residue. Furthermore, deletion of CXT1 led to attenuation of cryptococcal growth in a mouse model of infection, suggesting that the affected xylose residues are important for normal host-pathogen interactions. Cxt1p is the first glycosyltransferase with a defined role in C. neoformans capsule biosynthesis, and cxt1Delta is the only strain identified to date with structural alterations of the capsule polysaccharide GalXM.     

The physical properties of the capsular polysaccharides from Cryptococcus neoformans suggest features for capsule construction

The most distinctive feature of the human pathogenic fungus is a polysaccharide capsule that is essential for virulence and is composed primarily of glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). GXM mediates multiple deleterious effects on host immune function, yet relatively little is known about its physical properties. The average mass of Cryptococcus neoformans GXM from four antigenically different strains ranged from 1.7 to 7 x 10(6) daltons as calculated from Zimm plots of light-scattering data. GalXM was significantly smaller than GXM, with an average mass of 1 x 10(5) daltons. These molecular masses imply that GalXM is the most numerous polysaccharide in the capsule on a molar basis. The radius of gyration of the capsular polysaccharides ranged between 68 and 208 nm. Viscosity measurements suggest that neither polysaccharide altered fluid dynamics during infection since GXM behaved in solution as a polyelectrolyte and GalXM did not increase solution viscosity. Immunoblot analysis indicated heterogeneity within GXM. In agreement with this, scanning transmission electron microscopy of GXM preparations revealed a tangled network of two different types of molecules. Mass per length measurements from light scattering and scanning transmission electron microscopy were consistent and suggested GXM molecules self-associate. A mechanism for capsule growth is proposed based on the extracellular release and entanglement of GXM molecules.     

Capsular polysaccharides galactoxylomannan and glucuronoxylomannan from Cryptococcus neoformans induce macrophage apoptosis mediated by Fas ligand

The effects of capsular polysaccharides, galactoxylomannan (GalXM) and glucuronoxylomannan (GXM), from acapsular (GXM negative) and encapsulate strains of Cryptococcus neoformans were investigated in RAW 264.7 and peritoneal macrophages. Here, we demonstrate that GalXM and GXM induced different cytokines profiles in RAW 264.7 macrophages. GalXM induced production of TNF-alpha, NO and iNOS expression, while GXM predominantly induced TGF-beta secretion. Both GalXM and GXM induced early morphological changes identified as autophagy and late macrophages apoptosis mediated by Fas/FasL interaction, a previously unidentified mechanism of virulence. GalXM was more potent than GXM at induction of Fas/FasL expression and apoptosis on macrophages in vitro and in vivo. These findings uncover a mechanism by which capsular polysaccharides from C. neoformans might compromise host immune responses.     

Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule-structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the PROPSEARCH software classified Cas1p as a putative glycosyltransferase. Cas1p is a well-conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O-acetylation.     

Cryptococcus neoformans capsular polysaccharide component galactoxylomannan induces apoptosis of human T-cells through activation of caspase-8

The major virulence factor of Cryptococcus neoformans is its polysaccharide capsule composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoproteins. A variety of immunomodulating activities have been described for GXM and mannoproteins but little is known about possible interactions of GalXM with the immune system. In the present article, we investigate the effect of purified soluble GalXM on human T lymphocytes. The results indicate that, GalXM (i) can affect selected immune responses; (ii) causes significant impairment of T cell proliferation and increases interferon-gamma and interleukin-10 production; and (iii) induces apoptosis of T lymphocytes through activation of caspase-8 that terminates with fragmentation of DNA. These results are the first to suggest a role for GalXM in C. neoformans virulence by demonstrating that it can target human T cells, and that it may impair the development of an effective specific T cell response.     

Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans

The capsule of Cryptococcus neoformans is the most obvious virulence factor of this pathogenic yeast. The main capsule constituents are glucuronoxylomannans (GXM). Although several studies have focused on GXM composition and structure, very little is known about their genetics. To elucidate the relationship between the capsule structure and the pathophysiology of the cryptococcosis, genetic screening for mutant strains producing a structurally modified capsule was set up. Using monoclonal antibodies specific for different capsule sugar epitopes, we isolated strains with different mutated capsule structures (Cas mutants). According to their reactivities with various monoclonal antibodies, the mutants were classified into six groups (Cas1 to Cas6). One Cas2 mutant was used to clone the corresponding gene by complementation. This gene (USX1) encodes the previously identified UDP-xylose synthase. We demonstrated that it is necessary for both capsule xylosylation and C. neoformans virulence.     

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.     

Cas3p belongs to a seven-member family of capsule structure designer proteins

The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans. Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear. A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C. neoformans genome. This gene family is apparently specific in a subclass of the basidiomycete fungi. Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35Delta strains displayed a hypocapsular phenotype. The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone. Hence, these genes are all involved in assembly of the GXM structure in C. neoformans.     

Parallel beta-helix proteins required for accurate capsule polysaccharide synthesis and virulence in the yeast Cryptococcus neoformans

The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an alpha-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel beta-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.     

Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans

The virulence of the human pathogenic fungus Cryptococcus neoformans is regulated by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling cascade that promotes mating and the production of melanin and capsule. In this study, genes encoding homologs of the Saccharomyces cerevisiae low- and high-affinity phosphodiesterases, PDE1 and PDE2, respectively, were deleted in serotype A strains of C. neoformans. The resulting mutants exhibited moderately elevated levels of melanin and capsule production relative to the wild type. Epistasis experiments indicate that Pde1 functions downstream of the Galpha subunit Gpa1, which initiates cAMP-dependent signaling in response to an extracellular signal. Previous work has shown that the PKA catalytic subunit Pka1 governs cAMP levels via a negative feedback loop. Here we show that a pde1Delta pka1Delta mutant strain exhibits cAMP levels that are dramatically increased ( approximately 15-fold) relative to those in a pka1Delta single mutant strain and that a site-directed mutation in a consensus PKA phosphorylation site reduces Pde1 function. These data provide evidence that fluctuations in cAMP levels are modulated by both Pka1-dependent regulation of Pde1 and another target that comprise a robust negative feedback loop to tightly constrain intracellular cAMP levels.     

Role for Golgi reassembly and stacking protein (GRASP) in polysaccharide secretion and fungal virulence

Secretion of virulence factors is a critical mechanism for the establishment of cryptococcosis, a disease caused by the yeast pathogen Cryptococcus neoformans. One key virulence strategy of C. neoformans is the release of glucuronoxylomannan (GXM), a capsule-associated immune-modulatory polysaccharide that reaches the extracellular space through secretory vesicles. Golgi reassembly and stacking protein (GRASP) is required for unconventional protein secretion mechanisms in different eukaryotic cells, but its role in polysaccharide secretion is unknown. This study demonstrates that a C. neoformans functional mutant of a GRASP orthologue had attenuated virulence in an animal model of cryptococcosis, in comparison with wild-type (WT) and reconstituted cells. Mutant cells manifested altered Golgi morphology, failed to produce typical polysaccharide capsules and showed a reduced ability to secrete GXM both in vitro and during animal infection. Isolation of GXM from cultures of WT, reconstituted or mutant strains revealed that the GRASP orthologue mutant produced polysaccharides with reduced dimensions. The mutant was also more efficiently associated to and killed by macrophages than WT and reconstituted cells. These results demonstrate that GRASP, a protein involved in unconventional protein secretion, is also required for polysaccharide secretion and virulence in C. neoformans.     

Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans

Cryptococcus neoformans is a human pathogenic fungus with a capsule composed primarily of glucuronoxylomannan (GXM) that is important for virulence. Current views of GXM structure postulate a polymer composed of repeating mannose trisaccharide motifs bearing a single beta(1,2) glucuronic acid with variable xylose and O-acetyl substitutions to form six triads. GXM from different strains is notoriously variable in triad composition, but it is not known if the polymer consists of one or more motif-repeating units. We investigated the polymeric organization of GXM by using mass spectrometry to determine if its compositional motif arrangement was similar to that of bacterial capsular polysaccharides, namely, a polymer of a single repeating unit. The results were consistent with, and confirmatory for, the current view that the basic unit of GXM is a repeating mannose trisaccharide motif, but we also found evidence for the copolymerization of different GXM repeating units in one polysaccharide molecule. Analysis of GXM from isogenic phenotypic switch variants suggested structural differences caused by glucuronic acid positional effects, which implied flexibility in the synthetic pathway. Our results suggest that cryptococcal capsule synthesis is fundamentally different from that observed in prokaryotes and employs a unique eukaryotic approach, which theoretically could synthesize an infinite number of structural combinations. The biological significance of this capsule construction scheme is that it is likely to confer a powerful avoidance strategy for interactions with the immune system and phagocytic environmental predators. Consistent with this premise, the antigenic variation of a capsular epitope recognized by a nonprotective antibody was observed under different growth conditions.     

The Cryptococcus neoformans cap10 and cap59 mutant strains, affected in glucuronoxylomannan synthesis, differentially activate human dendritic cells

The human pathogen Cryptococcus neoformans causes meningo-encephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides: glucuronoxylomannan and galactoxylomannan. The presence of this polysaccharide capsule was previously shown to interfere with maturation of human dendritic cells (DCs), possibly by shielding cell-wall components from interacting with these host immune cells. Here we show that two mutant strains of C. neoformans , both lacking a visible capsule due to a defect in glucuronoxylomannan synthesis, differentially activate human monocyte-derived DCs. Cells from a cap59 mutant, but not of a cap10 mutant strain, induce maturation of DCs as indicated by an increase in the expression of the costimulatory molecules CD80 and CD86, and production of the cytokines interleukin (IL)-10, IL-12p40 and tumor necrosis factor α. Interestingly, cap59 mutant cells reassociated with a concentrated culture medium of wild-type C. neoformans had lost their capacity to induce DC maturation. Summarizing, our data suggest that glucuronoxylomannan confers properties to the capsule that protect the fungus against activation of DCs; however, the presence of intact glucuronoxylomannan is not an absolute requirement to prevent activation of DCs.     

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 ( AFR1 ) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1- overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1 Δ mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1 -overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans –microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.     

Characterization of pathogenic constituents of Cryptococcus neoformans strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 X 10(5) or 1 X 10(7) viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; a cell surface structure for responsible for resistance to intra- or extracellular killing and induction of the granulomatous lesion; a growth rate suitable for interacting with phagocytic elimination.     

Cryptococcus neoformans CAP59 (or Cap59p) is involved in the extracellular trafficking of capsular glucuronoxylomannan

Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.     

The cellular responses induced by the capsular polysaccharide of Cryptococcus neoformans differ depending on the presence or absence of specific protective antibodies

The capsule of Cryptococcus neoformans, the principal virulence factor of this fungus, is composed primarily of polysaccharide. The predominant component of the polysaccharide capsule is glucuronoxylomannan (GXM), a compound with potent immunoregulatory properties. GXM is bound and internalized by natural immune cells affecting innate and subsequent adaptive immune response. The cellular pattern recognition receptors involved in GXM binding include toll-like receptor (TLR)4, CD14, TLR2, CD18, Fc gamma receptor II (FcgammaRPi). This multiple cross-linking leads to a suppressive outcome that is arrested and even reversed by protective antibodies to GXM. This review analyzes the immunosuppressive effects induced by capsular material, considering its pattern recognition receptors, and dissects the mechanism of monoclonal antibody shifting to immunoactivation.     

Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans

The cell wall of pathogenic fungi such as Cryptococcus neoformans , provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild-type strains containing empty plasmid, but a complete absence in the i SEC6 strain. In addition, a green fluorescent protein–laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in i SEC6 strains. In contrast, i SEC6 strains retained normal growth at 37°C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans .     

Production of Extracellular Polysaccharides by CAP Mutants of Cryptococcus neoformans

The human pathogen Cryptococcus neoformans causes meningoencephalitis. The polysaccharide capsule is one of the main virulence factors and consists of two distinct polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM). How capsular polysaccharides are synthesized, transported, and assembled is largely unknown. Previously, it was shown that mutations in the CAP10, CAP59, CAP60, and CAP64 genes result in an acapsular phenotype. Here, it is shown that these acapsular mutants do secrete GalXM and GXM-like polymers. GXM and GalXM antibodies specifically reacted with whole cells and the growth medium of the wild type and CAP mutants, indicating that the capsule polysaccharides adhere to the cell wall and are shed into the environment. These polysaccharides were purified from the medium, either with or without anion-exchange chromatography. Monosaccharide analysis of polysaccharide fractions by gas-liquid chromatography/mass spectrometry showed that wild-type cells secrete both GalXM and GXM. The CAP mutants, on the other hand, were shown to secrete GalXM and GXM-like polymers. Notably, the GalXM polymers were shown to contain glucuronic acid. One-dimensional ¹H nuclear magnetic resonance confirmed that the CAP mutants secrete GalXM and also showed the presence of O-acetylated polymers. This is the first time it is shown that CAP mutants secrete GXM-like polymers in addition to GalXM. The small amount of this GXM-like polymer, 1 to 5% of the total amount of secreted polysaccharides, may explain the acapsular phenotype.Cryptococcus neoformans of the A (var. grubii [24]) and D (var. neoformans [36]) serotypes are the causative agents of cryptococcosis, of which the most common clinical form is meningoencephalitis. This disease is related to immunocompromised patients but can also occur in immunocompetent individuals (4, 19, 38). One of the main virulence factors is the polysaccharide capsule (2, 5, 17, 21, 27, 35). This capsule enables the yeast-like fungus to survive the harsh environment of the human body by using its immunomodulatory properties that enable immune evasion and by preventing killing through phagocytosis by macrophages (44, 45).The capsule consists of a low percentage of mannoproteins (46) and the polysaccharides glucuronoxylomannan (GXM) and galactoxylomannan (GalXM) in a mass ratio of about 10:1 (14, 16, 17). Little is known about the synthesis of GXM and GalXM and their transport toward the cell surface. A mutation in the Sec4/Rab8 GTPase homologue was recently shown to affect protein secretion as well as polysaccharide secretion and resulted in intracellular accumulation of vesicles containing GXM (51). From this and the fact that GXM has been detected in extracellular vesicles, it was proposed that polysaccharides are packaged in such vesicles to cross the cell wall to reach the extracellular environment (47).Mutation analysis has revealed four genes, called CAP10, CAP59, CAP60, and CAP64, which give an acapsular phenotype when inactivated (7, 9-13). The precise role of the encoded Cap proteins is unknown. Cap59 has been suggested to play a role in extracellular trafficking of multimeric forms of GXM molecules (26). Moreover, it may play a role in the assembly of GXM, since it shares homology with a mannosyltransferase (48). Like Cap59, Cap60 is a putative mannosyltransferase. Cap10 shares homology with a xylosyltransferase and therefore may also be involved in capsule assembly (34), like the recently identified xylosyltransferase encoded by CXT1 (33). This transferase has been shown to play a direct role in the synthesis of both of the capsular polysaccharides but is especially active in the addition of xyloses to the GalXM polysaccharide. CAP64 shares homology with so-called CAS genes, encoding proteins involved in O acetylation of GXM (40).Structural analysis has revealed a relatively clear picture of the buildup of the GXM and GalXM polysaccharides (14, 50) (Fig. ​(Fig.1).1). Some variability in the chemical structures of the capsular polysaccharides has been described, even within the capsule of a particular strain (40, 50). In addition, GalXM has been shown to also contain, besides galactopyranose, galactofuranose in trace amounts (1, 29). The two C. neoformans serotypes A and D are distinguished based on variation in the position of the different xylose residues in the GXM repeating unit (30). The structure of the GalXM repeating unit was analyzed by using a fraction of purified polysaccharides secreted in the medium by a mutant of the D serotype called the CAP67 mutant. This strain is mutated in the same gene as a serotype A CAP59 mutant. The number of xylose residues can vary from zero up to six within the GalXM repeating unit (Fig. ​(Fig.1)1) (50).Open in a separate windowFIG. 1.Chemical structure of GXM and GalXM monomers. Large strands of these monomers form polymers of up to 1 × 10⁶ to 7 × 10⁶ daltons for GXM and 1 × 10⁵ daltons for GalXM. Ratios vary between serotypes. Shown are serotype A GXM, Man 3/Xyl 2/GlcA 1, and GalXM, Gal 6/Man 4/Xyl 1.6 (shown are three xyloses). The degree of O acetylation is not shown. The picture is based on data from reference 3.So far, secreted polysaccharides in the medium of the serotype D CAP67 mutant and the corresponding serotype A CAP59 mutant have been analyzed (41, 50). It was shown that these mutants secrete GalXM but not GXM in the medium. However, it is shown here that these mutants, as well as the serotype A CAP10, CAP60, and CAP64 mutants, also secrete GXM-like polymers in addition to GalXM. Moreover, part of GalXM seems to contain glucuronic acid, supporting earlier findings (16, 49).     

Phenotypic heterogeneity in expression of epitopes in the Cryptococcus neoformans capsule

The opportunistic yeast Cryptococcus neoformans is surrounded by a polysaccharide capsule comprised primarily of glucuronoxylomannan (GXM). GXM is a key component of the antigenic character of the capsule. Expression of the epitope that allows for binding of mAbs that require O -acetylation of GXM for mAb recognition was greatly influenced by cell age, growth conditions and serotype. Yeast cells of serotype A grown in vitro under capsule induction conditions showed considerable cell-to-cell variability in binding of two O -acetyl-dependent mAbs, and such mAbs uniformly failed to bind to GXM that covers yeast buds. Expression of the O -acetyl-dependent epitope increased with cell age. In contrast, all serotype A cells harvested from brain tissue bound the same O -acetyl-dependent mAbs. The ability of the cryptococcal capsule to activate the complement cascade and bind C3 occurred uniformly over the surface of all yeast cells, including the bud. Finally, the cell-to-cell variability in binding of O -acetyl-dependent mAbs with strains of serotype A was not found with strains of serotype D; almost all cells of serotype D showed homogeneous binding of O -acetyl-dependent mAbs. These results indicate that variability in expression of antigenic epitopes by GXM should be considered in selection of mAbs used for immunodiagnosis or immunotherapy.