Fungal Mannan Research Articles

TRAF1 negatively regulates C-type lectin receptor-induced proinflammatory response to fungal infection

Abstract Fungal infections pose serious health threat worldwide, causing severe mucosal and systemic candidiasis in elderly people, AIDS patients and organ recipients. Through sensing fungal cell-wall components b-glucan and mannan, C-type lectin receptors (CLRs) dectin-1 and dectin-2/3 play pivotal role in the induction of anti-fungal innate and adaptive immune responses. However, the regulatory mechanisms of CLR signaling remain to be better understood. Indeed, our previous work demonstrated that the protein tyrosine phosphatase SHP-2 acts as a positive regulator of CLR-induced signaling, and thus plays a critical role in DCs to promote anti-fungal Th17 response. In this study, we found that fungus-elicited CLR signals are also stringently controlled by negative regulation. Upon C. albicans infection, TRAF1 was highly induced in skin, lung and kidney. Elevated TRAF1 expression was also detected in macrophages and DCs stimulated by dectin-1 and dectin-2/3 ligands, respectively. Mechanistically, TRAF1 acted as a feed-back negative regulator critically controlling the induction of proinflammatory genes, such as Cxcl1 and Tnf, in response to fungal infection. Consistently, TRAF1-deficient mice exhibited increased neutrophil-infiltration, highly efficient fungal eradication and ameliorated tissue damage, culminating on improved host defense and better survival. Taken together, this study identified a new feed-back regulatory mechanism by which CLRs regulate anti-fungal proinflammatory response.

Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo.

Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived β1,3-glucans contribute to the EPS matrix structure, while fungal mannan and β-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

Mannosylation in Candida albicans: role in cell wall function and immune recognition

The fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post-translational modifications being the addition of O- and N-linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O- and N-linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host-fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease.

Differential Virulence of Candida glabrata Glycosylation Mutants

The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about the attributes that allow this organism to cause disease or its interaction with the host immune system. However, in common with other fungi, the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such, it is likely to play an important role in mediating interactions and hence virulence. Here, we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2, and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyltransferases. Furthermore, we show that deletion of the C. glabrata Anp1, Mnn2, and Mnn11 mannosyltransferases directly affects the structure of the fungal N-linked mannan, in line with their predicted functions, and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared with wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the long term, a better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.

Role of the Mycobiome in Human Acute Graft-versus-Host Disease

A role for gut bacteria in the pathogenesis of graft-versus-host disease (GVHD) has been firmly established; however, the role of Candida spp, which form part of the mycobiome, remains unknown. In a homogenous group of patients who underwent allogeneic stem cell transplantation (SCT), we found a significant impact of Candida colonization on the occurrence of acute GVHD. Patients colonized with Candida spp developed significantly more grade II-IV acute GVHD compared with noncolonized patients (50% vs 32%; P = .03), as well as more gastrointestinal (GI)-GVHD (33% vs 19%; P = .05). Colonization with Candida spp was more frequent in patients bearing the loss-of-function polymorphism Y238X, which results in dectin-1 dysfunction, compared with patients with the wild-type allele (73% vs 31%; P = .002). There was no direct effect of dectin-1 dysfunction on acute GVHD, although it did influence the occurrence of GVHD indirectly through Candida colonization. The exact mechanism of GVHD induction by Candida spp colonization of the mucosa is unknown, but the link might prove to be the induction of Th 17/IL-23 responses through activation of pattern recognition receptors by fungal motifs, including β-d-glucan and mannans. These data indicate a role for the mycobiome in the pathogenesis of GVHD and suggest that altering the mycobiome by antifungal drugs can help ameliorate GI-GVHD. In addition, given that the genetic constitution of patients affects susceptibility to both Candida colonization and GVHD, whether identifying gene polymorphisms will facilitate personalized treatment of SCT recipients remains to be determined.

Dectin-1 and NOD2 mediate cathepsin activation in zymosan-induced arthritis in mice

Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis. The roles of CR3, TLR2, MyD88, NOD1, NOD2, Dectin-1 and Dectin-2 were tested in vivo in arthritis elicited by intra-articular injections of zymosan, the fungal cell wall components curdlan, laminarin and mannan, and the bacterial cell wall peptidoglycan. Dectin-1, and to a lesser extent Dectin-2, contributed to arthritis. TLR2, MyD88 and CR3 played non-essential roles. Observations based on injection of curdlan, laminarin or mannan supported the dominant role of the Dectin-1 pathway in the joint. We demonstrated differential roles for NOD1 and NOD2 and identified NOD2 as a novel and essential mediator of zymosan-induced arthritis. Together, Dectin-1 and NOD2 are critical, sentinel receptors in the arthritogenic effects of zymosan. Our data identify a novel role for NOD2 during inflammatory responses within joints.

The Candida Th17 response is dependent on mannan- and -glucan-induced prostaglandin E2

The fungus Candida albicans is a potent inducer of the T(h)17 response. Prostaglandin E2 (PGE2) is a strong pro-inflammatory mediator, which has proven to be essential for the T(h)17 response. In this study, we have investigated the role of PGE2 in the T(h)17 response induced by C. albicans in humans. PBMC were stimulated with C. albicans in the absence or presence of a non-steroidal anti-inflammatory drug (NSAID). In separate experiments, PGE2 or the prostlaglandin receptors agonists butaprost or misoprostol were added to the cells. PBMC were also stimulated with fungal components and small interfering RNA for mannose receptor (MR) was performed. PGE2 and cytokines were measured by ELISA or luminex, and the source of IL-17 production was determined using FACS analysis. Blocking Candida-induced PGE2 production by an NSAID resulted in decreased IL-17 and IL-22 production and inhibited expression of RAR-related orphan receptor gamma T mRNA. Furthermore, when PGE2 production was blocked, IL-6, IL-23 and IL-10 were decreased, while tumor necrosis factor α increased. Stimulation with PGE2 or E prostanoid (EP)2/EP4 agonists restored IL-17 production. Candida albicans mannan was the only fungal component that was able to directly induce PGE2 and silencing of the MR resulted in a reduction of Candida-induced PGE2. β-Glucan engagement of dectin-1 synergistically increased Toll-like receptor 2 (TLR2)-induced PGE2 production. These data provide evidence that PGE2 pathway is important for the T(h)17 response induced by C. albicans and that PGE2 is induced by the fungal components mannan and β-glucan that are recognized by the MR and the dectin-1/TLR2 pathway, respectively.

Fungal Zymosan and Mannan Activate the Cryopyrin Inflammasome

Some fungal species are opportunistic pathogens that can cause infection in people with compromised immune systems. Activation of caspase-1 and the subsequent secretion of mature interleukin (IL)-1beta is a major signaling pathway of the innate immune system, but how yeasts induce caspase-1 activation is unknown. We show here that stimulation of macrophages and dendritic cells with heat-killed Saccharomyces cerevisiae or the purified cell wall components zymosan and mannan induced caspase-1 activation and IL-1beta secretion when combined with ATP. Macrophages deficient for the inflammasome adaptor ASC were defective in caspase-1 activation and IL-1beta secretion, suggesting involvement of an ASC-dependent inflammasome. Indeed, caspase-1 activation was abrogated in macrophages lacking the NOD-like (NLR) protein Cryopyrin/Nalp3 and in wild type macrophages pretreated with the pannexin-1 inhibitor probenecid. IL-1beta secretion further required the Toll-like receptor (TLR) adaptors MyD88 and TRIF, and partially relied on TLR2. We previously showed that bacterial molecules such as lipopolysaccharide (LPS) and peptidoglycan induce activation of caspase-7 through the Cryopyrin inflammasome. Similarly, Cryopyrin and ASC were required for activation of caspase-7 in macrophages stimulated with zymosan or mannan and ATP. These results demonstrate that the conserved fungal components zymosan and mannan require ASC and Cryopyrin for caspase-1 activation and IL-1beta secretion and suggest an important role for the Cryopyrin inflammasome during fungal infections.

Calcium is not required for Immulectin-2 binding, but protects the protein from proteinase digestion

Mammalian C-type lectins are calcium-dependent carbohydrate-binding proteins. They serve as cell adhesion molecules in cell–cell interactions, or function as pattern-recognition receptors in innate immunity. Calcium is a direct ligand for carbohydrate binding in mammalian C-type lectins such as mannose-binding proteins and macrophage mannose receptor. In the tobacco hornworm Manduca sexta, a group of lectins named immulectins have been discovered. Each immulectin contains dual carbohydrate-recognition domains. Previously, we showed that immulectin-2 (IML-2) binds to a bacterial lipopolysaccharide, and agglutination of Escherichia coli cells by IML-2 is calcium dependent. In this study, we demonstrated that IML-2 bound to bacterial lipid A, smooth and rough mutants of lipopolysaccharide, lipoteichoic acid and peptidoglycan, as well as to fungal mannan and β-1, 3-glucan (laminarin and curdlan). Binding of IML-2 to microbial components was calcium independent, and was increased by addition of spermine, a polyamine. In addition, plasma IML-2 bound to mannan-agarose independent of calcium. But trypsin digestion of IML-2 was inhibited in the presence of calcium. Our results suggest that calcium is not required for IML-2 binding but protects IML-2 from trypsin digestion.

Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each with identical N-terminal tags remote from the ligand-binding surface. Although rat and mouse showed similar affinities for saccharide competitors, both differed markedly from the human protein. The human neck+CRD preferentially recognized N-acetyl-mannosamine, whereas the rat and mouse proteins showed greater affinity for myoinositol, maltose, and glucose. Although human neck+CRDs bound to maltosyl-agarose and fungal mannan, only rat and mouse neck+CRDs showed significant binding to maltosyl-Toyopearl beads, solid-phase maltosyl-albumin neo-glycoprotein, or the Phil82 strain of influenza A virus. Likewise, human SP-D dodecamers and trimeric subunits of full-length rat, but not full-length human SP-D trimers, bound to maltosyl-Toyopearl. Site-directed mutagenesis of the human neck+CRD demonstrated an important role of Asp324-Asp325 in the recognition of N-acetyl-mannosamine, and substitution of the corresponding murine sequence (Asn324-Asn325) conferred a capacity to interact with immobilized maltose. Thus, ligand recognition by human SP-D involves a complex interplay between saccharide presentation, the valency of trimeric subunits, and species-specific residues that flank the primary carbohydrate binding site.

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose

We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.

Stimulation, inhibition and death of macrophages infected with Trichophyton rubrum

Trichophyton rubrum is the most common pathogen causing dermatophytosis, accounting for approximately 80% of the reported cases of onychomycosis. Since 90% of the chronic dermatophyte infections are caused by T. rubrum, it is likely that this pathogen must have evolved mechanisms that evade or suppress cell-mediated immunity. Several reports have highlighted the participation of phagocytes in the immune defense against fungi; however, few studies have addressed the role of these cells in dermatophytosis. In this study, we investigated the interactions of resident and peritoneal macrophages with T. rubrum. We show here that the interaction of T. rubrum conidia with resident macrophages results in the production of TNF-α and IL-10 but not IL-12 and nitric oxide. Infected macrophages down-regulated the expression of co-stimulatory molecules (CD80 and CD54). We also show that phagocytosis of T. rubrum conidia is inhibited by the addition of fungal exoantigens or mannan. Cytotoxicity assays indicated that after 8 h of conidia ingestion macrophage viability decreased drastically. Electron microscopy revealed that the ingested conidia grow and differentiate into hyphae inside macrophages leading to rupture of the macrophage membrane.

Saccharomyces cerevisiae- and Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner.

The cytokine-inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll-like receptors (TLRs). Tumor necrosis factor alpha (TNF-alpha) production by monocytes was markedly induced in a dose-dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 microg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF-alpha production than respective preparations from the hyphal form. Only slight TNF-alpha production was induced by the S. cerevisiae glucan. The TNF-alpha production triggered by reference LPS and purified fungal mannans required the presence of LPS-binding protein (LBP), and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan-LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

Synthesis and conformational analysis of methyl 2‐O‐(α‐D‐Mannopyranosyl)‐α‐D‐mannopyranoside

AbstractNMR experiments such as steady state NOE experiments and spin lattice 1H relaxation time measurements were performed on the synthetic disaccharide 10 that constitutes part of the polysaccharide backbone in fungal mannans. The spectro‐scopic data were compared with a theoretical model that is based upon the HSEA force field and statistical mechanics, taking into account the flexibility around the glycosidic linkage. Potential energy calculations were performed with a modified version of the GESA program in order to derive the global minimum conformation of disaccharide 10 and to calculate ensemble average NOE and T1 values by averaging all energetically accessible conformational microstates, using a normalized Boltzmann distribution function. The fit between experimental and theoretical results improved significantly when we applied a dynamic model instead of a single state model based solely on the global minimum conformation of 10. Efficient synthesis of 10 was achieved by a recently published protocol for the in situ activation of thioglycosides.

Pathogenesis of candidiasis. Immunosuppression by cell wall mannan catabolites.

Candida albicans cell wall mannan polysaccharide has an ability to negatively influence cell-mediated immune function. We have attempted to identify the mechanism of this phenomenon by testing the modulatory effects of isolated mannan and the chemical catabolites of mannan on cell-mediated immune function in vitro. We have determined that mannan isolated by complexation with cetyltrimethylammonium bromide (CTAB) is more antigenic than mannan isolated by precipitation with copper and that CTAB mannan does not inhibit lymphoproliferation stimulated by another antigen. We have also determined that oligosaccharides of three sizes, derived by chemical catabolism of CTAB mannan, are not antigenic, but instead are immunoinhibitory. Immunoinhibition does not involve interference with the mitogenic activity of interleukin 2. A similar occurrence of oligosaccharides may be produced by catabolism of mannan in vivo as evidenced by the presence of oligosaccharides of similar size in cell-free supernatant fluids derived from mononuclear leukocytes incubated with tritiated mannan. We propose that catabolites of fungal mannan may contribute significantly to suppression of cell-mediated immunity in candidiasis.

Application of mild acetolysis to confirm the comb-like structure of cell wall mannan from Pichia pastoris IFO0948 strain.

In order to confirm the chemical structure of the mannan of Pichia pastoris IFO 0948 strain, which was assumed to possess a highly branched comb-like structure, a sequential degradation procedure involving partial acid hydrolysis followed by acetolysis under mild conditions was employed. This procedure can widely be adopted as a method for determining the structural shape of fungal mannans possessing a highly branched structure, either comb-like or tree-like, without application of enzymolytic degradation.

Receptor-mediated clearance of Aspergillus galactomannan.

The fate of radiolabeled Aspergillus fumigatus galactomannan was studied after intravenous injection into rabbits and rats. At 1 hr, the liver contained 35% of the injected dose in rabbits and 30% in rats. Excretion of galactomannan into the urine, measured in rabbits, was another major catabolic route and accounted for 35% of the dose by 24 hr. Immunization of rabbits increased hepatic uptake and decreased urinary excretion. Hepatic uptake in unimmunized rats could be decreased by Saccharomyces cerevisiae mannan, alpha-methyl mannoside, and N-acetylglucosamine, known inhibitors of the macrophage mannose receptor. Autoradiography showed hepatic radiolabeled galactomannan to be concentrated in Kupffer cells, which express the mannosyl receptor for glycoproteins. Macrophage mannosyl receptors may constitute a general mechanism for clearing fungal mannans from the bloodstream.

Isolation and Purification of a Peptido-Rhamnomannan from the Yeast Form of Sporothrix Schenckii. Structural and Immunochemical Studies

Abstract A peptido-rhamnomannan was isolated from the cells and culture medium of the human pathogenic fungus Sporothrix schenckii, grown in the yeast phase. It was purified by precipitation of its borate complex with hexadecyltrimethylammonium bromide and then by DEAE-Sephadex chromatography. The sugars in the polysaccharide were identified as d-mannose (50%) and l-rhamnose (33%). The amino acid composition of the peptide resembled that of the peptide moiety of other fungal peptido-polysaccharides. The polymer was almost entirely (91%) precipitated by concanavalin A and the ratio of mannose to rhamnose in the precipitate was unchanged, showing that the sugars are components of the same polymer. Methylation analysis and partial acid hydrolysis showed that the basic structure of the polysaccharide is a peptido-mannan to which numerous l-rhamnose residues are attached. About 60% of the rhamnose residues could be preferentially removed by mild acid hydrolysis. The peptido-rhamnomannan precipitated with the sera of individuals (3) with sporotrichosis. Sera from a small number of patients with other fungal diseases and from normal individuals did not precipitate within the sensitivity limits of the method. The specificity of two of the sera were examined in more detail by studying their cross-reactions with other fungal mannans.