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.     

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.     

Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence

The polysaccharidic capsule is the main virulence factor of Cryptococcus neoformans. It primarily comprised of two polysaccharides: glucuronoxylomannan (GXM, 88% of the capsule mass) and galactoxylomannan (GalXM, 7% of the capsule mass). We constructed a large collection of mutant strains in which genes potentially involved in capsule biosynthesis were deleted. We used a new post-genomic approach to study the virulence of the strains. Primers specific for unique tags associated with the disruption cassette were used in a real-time PCR virulence assay to measure the fungal burden of each strain in different organs of mice in multi-infection experiments. With this very sensitive assay, we identified a putative UDP-glucose epimerase (Uge1p) and a putative UDP-galactose transporter (Ugt1p) essential for C. neoformans virulence. The uge1Delta and ugt1Delta strains are temperature sensitive and do not produce GalXM but synthesize a larger capsule. These mutant strains (GalXM negative, GXM positive) are not able to colonize the brain even at the first day of infection whereas GXM-negative strains (GalXM positive) can still colonize the brain, although less efficiently than the wild-type strain.     

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.     

Unexpected diversity in the fine specificity of monoclonal antibodies that use the same V region gene to glucuronoxylomannan of Cryptococcus neoformans

Most mAbs to the capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans are generated from the same VH and VL gene families. Prior Ab studies have assessed protective efficacy, Id structure and binding to capsular polysaccharides, and peptide mimetics. These data have been interpreted as indicating that most mAbs to GXM have the same specificity. A new approach to Ab specificity analysis was investigated that uses genetic manipulation to generate C. neoformans variants with structurally different capsules. C. neoformans mutants expressing GXM with defective O-acetylation were isolated and complemented by the C. neoformans gene CAS1, which is necessary for the O-acetylation of GXM. The mAbs exhibited differences in their binding to the GXM from these mutant strains, indicating previously unsuspected differences in specificity. Analysis of three closely related IgMs revealed that one (mAb 12A1) bound to an epitope that did not require O-acetylation, another (mAb 21D2) was inhibited by O-acetylation, and the third (mAb 13F1) recognized an O-acetylation-dependent conformational epitope. Furthermore, an IgG Ab (mAb 18B7) in clinical development retained binding to de-O-acetylated polysaccharide; however, greater binding was observed to O-acetylated GXM. Our findings suggest that microbial genetic techniques can provide a new approach for epitope mapping of polysaccharide-binding Abs and suggest that this method may applicable for studying the antigenic complexity of polysaccharide Ags in other capsulated microorganisms.     

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.     

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.     

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.     

Self-aggregation of Cryptococcus neoformans capsular glucuronoxylomannan is dependent on divalent cations

The capsular components of the human pathogen Cryptococcus neoformans are transported to the extracellular space and then used for capsule enlargement by distal growth. It is not clear, however, how the glucuronoxylomannan (GXM) fibers are incorporated into the capsule. In the present study, we show that concentration of C. neoformans culture supernatants by ultrafiltration results in the formation of highly viscous films containing pure polysaccharide, providing a novel, nondenaturing, and extremely rapid method to isolate extracellular GXM. The weight-averaged molecular mass of GXM in the film, determined using multiangle laser light scattering, was ninefold smaller than that of GXM purified from culture supernatants by differential precipitation with cetyl trimethyl ammonium bromide (CTAB). Polysaccharides obtained either by ultrafiltration or by CTAB-mediated precipitation showed different reactivities with GXM-specific monoclonal antibodies. Viscosity analysis associated with inductively coupled plasma mass spectrometry and measurements of zeta potential in the presence of different ions implied that polysaccharide aggregation was a consequence of the interaction between the carboxyl groups of glucuronic acid and divalent cations. Consistent with this observation, capsule enlargement in living C. neoformans cells was influenced by Ca(2+) in the culture medium. These results suggest that capsular assembly in C. neoformans results from divalent cation-mediated self-aggregation of extracellularly accumulated GXM molecules.     

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.     

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.     

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.     

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.     

Radiological studies reveal radial differences in the architecture of the polysaccharide capsule of Cryptococcus neoformans

The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans is an important virulence factor, but relatively little is known about its architecture. We applied a combination of radiological, chemical, and serological methods to investigate the structure of this polysaccharide capsule. Exposure of C. neoformans cells to gamma radiation, dimethyl sulfoxide, or radiolabeled monoclonal antibody removed a significant part of the capsule. Short intervals of gamma irradiation removed the outer portion of the cryptococcal capsule without killing cells, which could subsequently repair their capsules. Survival analysis of irradiated wild-type, acapsular mutant, and complemented mutant strains demonstrated that the capsule contributed to radioprotection and had a linear attenuation coefficient higher than that of lead. The capsule portions remaining after dimethyl sulfoxide or gamma radiation treatment were comparable in size, 65 to 66 microm3, and retained immunoreactivity for a monoclonal antibody to glucuronoxylomannan. Simultaneous or sequential treatment of the cells with dimethyl sulfoxide and radiation removed the remaining capsule so that it was not visible by light microscopy. The capsule could be protected against radiation by either of the free radical scavengers ascorbic acid and sorbitol. Sugar composition analysis of polysaccharide removed from the outer and inner parts of the capsule revealed significant differences in glucuronic acid and xylose molar ratios, implying differences in the chemical structure of the constituent polysaccharides. Our results provide compelling evidence for the existence of two zones in the C. neoformans capsule that differ in susceptibility to dimethyl sulfoxide and radiation and, possibly, in packing and composition.     

Glucuronoxylomannan-mediated interaction of Cryptococcus neoformans with human alveolar cells results in fungal internalization and host cell damage

Infection by Cryptococcus neoformans begins with inhalation of infectious propagules. Fungi reach the lung tissue and interact with epithelial cells in a crucial but poorly understood process. In this study, the interaction of C. neoformans with the human alveolar epithelial cell lineage A549 was investigated, focusing on the relevance of the capsular polysaccharide in this process. The association of encapsulated strains with A549 cells was significantly inhibited by a monoclonal antibody to glucuronoxylomannan (GXM), a major component of the cryptococcal capsule. A purified preparation of GXM produced similar results, suggesting the occurrence of surface receptors for this polysaccharide on the surface of alveolar cells. A549 cells were in fact able to bind soluble GXM, as confirmed by indirect immunofluorescence analysis using the anti-polysaccharide antibody. C. neoformans is internalized after GXM-mediated interaction with A549 cells in a process that culminates with death of host cells. Our results suggest that C. neoformans can use GXM for attachment to alveolar epithelia, allowing the fungus to reach the intracellular environment and damage host cells through still uncharacterized mechanisms.     

Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.     

Glucuronoxylomannan from Cryptococcus neoformans down-regulates the enzyme 6-phosphofructo-1-kinase of macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.     

Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.     

Polysaccharides, mimotopes and vaccines for fungal and encapsulated pathogens.

Vaccination is a rational alternative to treatment for Cryptococcus neoformans infections, as these infections are currently intractable in immunocompromised (including HIV-infected) individuals. Vaccines composed of the cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), the key C. neoformans virulence factor, elicit protective antibodies in mice, although deleterious antibodies can also be induced. By contrast, polysaccharides are poor immunogens in HIV-infected humans and others with B-cell defects. Peptide mimotopes of GXM can induce protective immunity to C. neoformans in mice, however, our knowledge of the mechanisms of mimotope-induced protection is incomplete and further work is needed if polysaccharide- or mimotope-based vaccines are to be used to manage C. neoformans infection.