GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling.

Cryptococcus spp. has a polysaccharide capsule composed of glucuronoxylomannan-GXM, a major virulence factor that can prevent the recognition of fungi by immune cells. Chimeric Antigen Receptor (CAR) redirects T cells to target Cryptococcus spp. as previously demonstrated by a CAR specific to GXM, GXMR-CAR. The current study evaluated the strength of the signal transduction triggered by GXMR-CAR, composed of a distinct antigen-binding domain sourced from a single-chain variable fragment (scFv). GXM-specific scFv derived from mAbs 2H1 and 18B7, 2H1-GXMR-CAR and 18B7-GXMR-CAR, respectively, were designed to express CD8 molecule as hinge/transmembrane, and the costimulatory molecule CD137 (4-1BB) coupled to CD3ζ. The 2H1-GXMR-CAR or 18B7-GXMR-CAR Jurkat cells recognized soluble GXM from C. gattii and C. neoformans, and the levels of IL-2 released by the modified cells did not differ between the GXMR-CAR constructs after exposure to Cryptococcus spp. 18B7-GXMR-CAR triggered tonic signaling was more pronounced in modified Jurkat cells, and a protein kinase inhibitor of the Src family (dasatinib) significantly reduced GXMR-CAR tonic signaling and inhibited cell activation against ligands. 18B7 scFv showed a structural modification of the variable heavy (VH) chain that clarified the difference in the strength of tonic signaling and the level of cell activation between 2H1-GXMR-CAR and 18B7-GXMR-CAR. GXMR-CAR constructs induced T-cell activation against clinical isolates of Cryptococcus spp. and serum from patients with cryptococcosis induced high levels of IL-2, mainly in cells modified with 18B7-GXMR-CAR. Thus, 18B7-GXMR-CAR and 2H1-GXMR-CAR mediated T cell activation against Cryptococcus spp. and 18B7 and 2H1 scFv influenced the strength of tonic signaling.

2H1-GXMR-CAR and 18B7-GXMR-CAR are efficiently expressed on the cell surface;2H1-GXMR-CAR and 18B7-GXMR-CAR redirected T cells toward the ligands;18B7-GXMR-CAR provided highest levels of tonic signaling;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR.

Calcium Binding Protein Ncs1 Is Calcineurin Regulated in Cryptococcus neoformans and Essential for Cell Division and Virulence.

Intracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signaling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+ binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and nonstress conditions, as the ncs1Δ mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to the case with Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ), and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1 deficiency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data show that Ncs1 has a significant role as a Ca2+ sensor in C. neoformans, working with calcineurin to regulate Ca2+ homeostasis and, consequently, promote fungal growth and virulence.IMPORTANCECryptococcus neoformans is the major cause of fungal meningitis in HIV-infected patients. Several studies have highlighted the important contributions of Ca2+ signaling and homeostasis to the virulence of C. neoformans Here, we identify the cryptococcal ortholog of neuronal calcium sensor 1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression, and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signaling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.

Polysaccharide diversity in VNI isolates of Cryptococcus neoformans from Roraima, Northern Brazil.

Species of the Cryptococcus genus comprise environmental, encapsulated fungal pathogens that cause lethal meningitis in immunosuppressed individuals. In humans, fungal uptake of hypocapsular Cryptococcus by macrophages was associated with high fungal burden in the cerebrospinal fluid and long-term patient survival. On the basis of the key role of the cryptococcal capsule in disease, we analyzed the diversity of capsular structures in 23 isolates from pigeon excreta collected in the cities of Boa Vista, Bonfim and Pacaraima, in the state of Roraima (Northern Brazil). All isolates were identified as Cryptococcus neoformans (VNI genotype) by MALDI-TOF mass spectrometry. Through a combination of fluorescence microscopy, flow cytometry, ELISA and spectrophotometric methods, each isolate was characterized at the phenotypical level, which included measurements of growth rates at 30 and 37 °C, pigmentation, cell body size, capsular dimensions, serological reactivity, urease production and ability to produce extracellular glucuronoxylomannan (GXM), the main capsular component of C. neoformans. With the exception of melanization, a formidable diversity was observed considering all parameters tested in our study. Of note, hyper and hypo producers of GXM were identified, in addition to isolates with hyper and hypo profiles of reactivity with a polysaccharide-binding monoclonal antibody. Capsular dimensions were also highly variable in the collection of isolates. Extracellular GXM production correlated positively with capsular dimensions, urease activity and cell size. Unexpectedly, GXM concentrations did not correlate with serological reactivity with the cryptococcal capsule. These results reveal a high diversity in the ability of environmental C. neoformans to produce capsular components, which might impact the outcome of human cryptococcosis.

Rheological properties of cryptococcal polysaccharide change with fiber size, antibody binding and temperature.

Aim:Cryptococcus neoformans is the major agent of cryptococcosis. The main virulence factor is the polysaccharide (PS) capsule. Changes in cryptococcal PS properties have been poorly elucidated. Materials & methods: We analyzed the mechanical properties of secreted PS and intact capsules, using dynamic light scattering and optical tweezers. Results: Storage and loss moduli showed that secreted PS behaves as a viscoelastic liquid, while capsular PS behaves as a viscoelastic solid. The secreted PS remains as a viscoelastic fluid at different temperatures with thermal hysteresis after 85°C. Antibody binding altered the viscoelastic behavior of both secreted and capsular PS. Conclusion: Deciphering the mechanical aspects of these structures could reveal features that may have consequences in novel therapies against cryptococcosis.

Characterization of the 6-O-acetylated lipoglucuronomannogalactan a novel Cryptococcus neoformans cell wall polysaccharide.

Glucuronoxylomannogalactans (GXMGals) are characteristic capsular polysaccharides produced by the opportunistic fungus C. neoformans, which are implicated in cryptococcal virulence, via impairment of the host immune response. We determined for the first time the structure of a lipoglucuronomannogalactan (LGMGal), isolated from the surface of a mutant C. neoformans carrying a deletion in the UDP-GlcA decarboxylase gene. Monosaccharide composition and methylation analyses, as well as nuclear magnetic resonance spectroscopy were employed in discerning the structure. Our results show that the polysaccharide structure of the LGMGal differs from GXMGal by the absence of xylose and 2-O-acetylated mannose residues. LGMGal consists of a galactan main chain -[-6-α-Gal-]-, where every second Gal residue is substituted at O-3 with an oligosaccharide α-Man6OAc-3-α-Man-4-(ß-GlcA-3)-ß-Gal-; components in italic being non-stoichiometric. The substitution rate of ß-Galp units by GlcpA is 35%. Additionally, we determined that the glycolipid anchor of the LGMGal is based on an myo-inositol phosphoceramide composed of C18-phytosphingosine and monohydroxylated lignoceric acid (2OHC24:0 fatty acid).

The Overlooked Glycan Components of the Cryptococcus Capsule.

Pathogenic species of Cryptococcus kill approximately 200,000 people each year. The most important virulence mechanism of C. neoformans and C. gattii, the causative agents of human and animal cryptococcosis, is the ability to form a polysaccharide capsule. Acapsular mutants of C. neoformans are avirulent in mice models of infection, and extracellularly released capsular polysaccharides are deleterious to the immune system. The principal capsular component in the Cryptococcus genus is a complex mannan substituted with xylosyl and glucuronyl units, namely glucuronoxylomannan (GXM). The second most abundant component of the cryptococcal capsule is a galactan with multiple glucuronyl, xylosyl, and mannosyl substitutions, namely glucuronoxylomannogalactan (GXMGal). The literature about the structure and functions of these two polysaccharides is rich, and a number of comprehensive reviews on this topic are available. Here, we focus our discussion on the less explored glycan components associated with the cryptococcal capsule, including mannoproteins and chitin-derived molecules. These glycans were selected for discussion on the basis that i) they have been consistently detected not only in the cell wall but also within the cryptococcal capsular network and ii) they have functions that impact immunological and/or pathogenic mechanisms in the Cryptococcus genus. The reported functions of these molecules strongly indicate that the biological roles of the cryptococcal capsule go far beyond the well-known properties of GXM and GXMGal.

Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes.

Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and C. gattii genotype differentiation. However, Vitek MS, one of the commercial MALDI-TOF MS instruments, has not been yet been evaluated for this purpose. Thus, we constructed an in-house database with reference strains belonging to the different C. neoformans (VNI, VNII, VNIII, and VNIV) and C. gattii (VGI, VGII, VGIII, and VGIV) major molecular types by using the software Saramis Premium (bioMérieux, Marcy-l'Etoile, France). Then, this new database was evaluated for discrimination of the different genotypes. Our in-house database provided correct identification for all C. neoformans and C. gattii genotypes; however, due to the intergenotypic mass spectral similarities, a careful postanalytic evaluation is necessary to provide correct genotype identification.

Peeling the onion: the outer layers of Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that is ubiquitous in the environment. It causes a deadly meningitis that is responsible for over 180,000 deaths worldwide each year, including 15% of all AIDS-related deaths. The high mortality rates for this infection, even with treatment, suggest a need for improved therapy. Unique characteristics of C. neoformans may suggest directions for drug discovery. These include features of three structures that surround the cell: the plasma membrane, the cell wall around it, and the outermost polysaccharide capsule. We review current knowledge of the fundamental biology of these fascinating structures and highlight open questions in the field, with the goal of stimulating further investigation that will advance basic knowledge and human health.

Distribution of the O-acetyl groups and ß-galactofuranose units in galactoxylomannans of the opportunistic fungus Cryptococcus neoformans.

Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that ß-galactofuranose (ß-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.

Fisetin as a promising antifungal agent against Cryptocococcus neoformans species complex.

AIMS: The aim of this study was to investigate the mechanisms of action of fisetin, a flavonol with antifungal activity previously evaluated against the Cryptococcus neoformans species complex. METHODS AND RESULTS: Ergosterol content and flow cytometry analysis were determined for the C. neoformans species complex in the presence of fisetin and ultrastructural analysis of morphology was performed on Cryptococcus gattii and C. neoformans. Decrease in the total cellular ergosterol content after exposure to fisetin ranged from 25·4% after exposure to 128 µg ml(-1) to 21·6% after exposure to 64 µg ml(-1) of fisetin compared with the control (without fisetin). The fisetin effects obtained with flow cytometry showed metabolic impairment, and alterations in its normal morphology caused by fisetin in C. neoformans cells were verified using scanning electron microscopy. CONCLUSIONS: Fisetin is a compound that acts in the biosynthesis of ergosterol. Flow cytometry showed that fisetin reduced viability of the metabolically active cells of C. gattii, while morphological changes explain the action of fisetin in inhibiting growth of these fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the idea that fisetin may represent a good starting point for the development of future therapeutic substances for cryptococcosis.

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67.

Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal-host relationship is discussed.

Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen.

Effects of Pimenta pseudocaryophyllus (Gomes) L. R. Landrum, on melanized and non-melanized Cryptococcus neoformans.

In the present study, the in vitro susceptibility and capsular width from both melanized and non-melanized Cryptococcus neoformans cells in the presence of Pimenta pseudocaryophyllus crude extract were determined. The results were compared with those obtained for voriconazole and amphotericin B. Melanization was obtained in minimal medium broth with the addition of L-dopa, and the antifungal susceptibility tests were performed using the broth microdilution method. Capsular width of 30 cells of each one of the isolates in medium with crude extracts of P. pseudocaryophyllus or voriconazole or amphotericin B at a concentration corresponding to 0.5 times the minimal inhibitory concentration (MIC) was measured, and the mean was calculated. The MICs and minimal fungicidal concentrations (MFCs) for plant extract and voriconazole were identical for both melanized and non-melanized C. neoformans isolates, but for amphotericin, the MFCs for melanized cells were up to 8 times higher than for non-melanized cells. The capsular width of C. neoformans cells was smaller (p < 0.001) in the presence crude extract of P. pseudocaryophyllus and of voriconazole regardless melanization. The findings of capsule alterations of C. neoformans verified in this study provide fertile ways for future research into the effects of antifungal agents on the pathogenesis of cryptococcosis.

Chitin-like molecules associate with Cryptococcus neoformans glucuronoxylomannan to form a glycan complex with previously unknown properties.

In prior studies, we demonstrated that glucuronoxylomannan (GXM), the major capsular polysaccharide of the fungal pathogen Cryptococcus neoformans, interacts with chitin oligomers at the cell wall-capsule interface. The structural determinants regulating these carbohydrate-carbohydrate interactions, as well as the functions of these structures, have remained unknown. In this study, we demonstrate that glycan complexes composed of chitooligomers and GXM are formed during fungal growth and macrophage infection by C. neoformans. To investigate the required determinants for the assembly of chitin-GXM complexes, we developed a quantitative scanning electron microscopy-based method using different polysaccharide samples as inhibitors of the interaction of chitin with GXM. This assay revealed that chitin-GXM association involves noncovalent bonds and large GXM fibers and depends on the N-acetyl amino group of chitin. Carboxyl and O-acetyl groups of GXM are not required for polysaccharide-polysaccharide interactions. Glycan complex structures composed of cryptococcal GXM and chitin-derived oligomers were tested for their ability to induce pulmonary cytokines in mice. They were significantly more efficient than either GXM or chitin oligomers alone in inducing the production of lung interleukin 10 (IL-10), IL-17, and tumor necrosis factor alpha (TNF-α). These results indicate that association of chitin-derived structures with GXM through their N-acetyl amino groups generates glycan complexes with previously unknown properties.

Presencia de Cryptococcus neoformans en excretas de palomas urbanas en San Miguel de Tucumán - Argentina / Presence of Cryptococcus neoformans in urban pigeons excreta from San Miguel de Tucumán – Argentina

Con la finalidad de detectar la presencia del complejo Cryptococcus neoformans en excretas de palomas (Columba livia) que habitan los espacios públicos del perímetro urbano de San Miguel de Tucumán, Argentina, se recogieron 100 muestras de 5 localizaciones diferentes. Como medio de aislamiento se empleó Agar-Semillas de Níger y las levaduras fueron identificadas mediante la presencia de cápsula, prueba de la ureasa, producción de fenol-oxidasa, asimilación de carbohidratos y crecimiento a 37°C. La especie fue determinada usando el medio Canavanina-Glicina-Azulde bromotimol. C. neoformans var. neoformans fue aislado en todas las 55 muestras de las áreas estudiadas, reflejando una amplia distribución en la zona céntrica de la ciudad preferentemente en excretas secas acumuladas. La sensibilidad in vitro frente a Fluconazol, Itraconazol, Anfotericina B y Voriconazol fue determinada por el método de difusión en placa usando tabletas Neo-Sensitabs. Todos los aislamientos fueron sensibles a los antifúngicos testeados.

With the purpose of detecting the presence of Cryptococcus neoformans complex in excreta of pigeons inhabitating public spaces belonging to the urban perimeter of San Miguel de Tucumán, Argentina, one hundred samples were collected from five different locations. Niger seed agar plates were used as means of isolation and yeasts were identified through the presence of capsule, urease test, fenoloxidase production, carbohydrate assimilation and growth at 37ºC. Species was determined with the CGB method. C. neoformans var. neoformans was isolated in all 55 samples of the studied areas showing a wide distribution downtown, mainly in dried excreta settlings. In vitro sensibility in front of Fluconazol, Itraconazol, Anfotericina B and Voriconazol was fixed by means of the diffusion method by using Neo-Sensitabs tablet. All isolations proved to be sensible to the tested antifungal.

Cryptococcus neoformans cryoultramicrotomy and vesicle fractionation reveals an intimate association between membrane lipids and glucuronoxylomannan.

Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.

Role for chitin and chitooligomers in the capsular architecture of Cryptococcus neoformans.

Molecules composed of beta-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.

The still obscure attributes of cryptococcal glucuronoxylomannan.

Glucuronoxylomannan (GXM) is the major capsular polysaccharide of Cryptococcus neoformans. It is essential for fungal virulence and causes a number of deleterious effects to host cells. During the last decades, most of the experimental models designed to study the roles of GXM during cryptococcal infection were based on the stimulation of animal cells. This most commonly involved macrophages or other effector cells, with polysaccharide fractions obtained by precipitation with cationic detergents. More recently, it has been demonstrated that GXM interferes with the physiological state of other target cells, such as the epithelium. In addition, recent studies indicate that the structure of the polysaccharide and, consequently, its functions vary according with the method used for its purification. This raises questions as to what is native GXM and the significance of prior studies. In this paper, we discuss some of the aspects of GXM that are still poorly explored in the current literature, including the relevance of the polysaccharide in the interaction of cryptococci with non-phagocytic cells and the relationship between its structure and biological activity.

Structural and functional properties of the Trichosporon asahii glucuronoxylomannan.

The virulence attributes of Trichosporon asahii are virtually unknown, despite its growing relevance as causative agent of superficial and invasive diseases in humans. Glucuronoxylomannan (GXM) is a well described virulence factor of pathogenic species in the Cryptococcus genus. GXM is also produced by species of the Trichosporon genus, and both polysaccharides share antigenic determinants, but unlike cryptococcal GXM, relatively little work has been done on trichosporal GXMs. In this study, we analyzed structural and functional aspects of GXM produced by T. asahii and compared them to the properties of the cryptococcal polysaccharide. Trichosporal and cryptococcal GXM shared antigenic reactivity, but the former polysaccharide had smaller effective diameter and negative charge. GXM anchoring to the cell wall was perturbed by dimethylsulfoxide and required interactions of chitin-derived oligomers with the polysaccharide. GXM from T. asahii supernatants are incorporated by acapsular mutants of Cryptococcus neoformans, which renders these cells more resistant to phagocytosis by mouse macrophages. In summary, our results establish that despite similarities in cell wall anchoring, antigenic and antiphagocytic properties, trichosporal and cryptococcal GXMs manifest major structural differences that may directly affect polysaccharide assembly at the fungal surface.

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.