Eliana Barreto-Bergter

An α-Glucan of Pseudallescheria boydii Is Involved in Fungal Phagocytosis and Toll-like Receptor Activation

The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an α-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that α-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked α-d-Glcp residues substituted at position 6 with α-d-Glcp branches. Soluble α-glucan, but not β-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when α-glucan from conidial surface was removed by enzymatic treatment with α-amyloglucosidase, thus indicating an essential role of α-glucan in P. boydii internalization by macrophages. α-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with α-amyloglucosidase. Finally, α-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and α-glucan could be a target for intervention during fungal infections.

An α-glucan of Pseudallescheria boydii is involved in fungal phagocytosis and TLR activation

The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an α-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that α-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked α-d-Glcp residues substituted at position 6 with α-d-Glcp branches. Soluble α-glucan, but not β-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when α-glucan from conidial surface was removed by enzymatic treatment with α-amyloglucosidase, thus indicating an essential role of α-glucan in P. boydii internalization by macrophages. α-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with α-amyloglucosidase. Finally, α-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and α-glucan could be a target for intervention during fungal infections.

Structure and biological functions of fungal cerebrosides

Ceramide monohexosides (CMHs, cerebrosides) are glycosphingolipids composed of a hydrophobic ceramide linked to one sugar unit. In fungal cells, CMHs are very conserved molecules consisting of a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids, and a carbohydrate portion consisting of one residue of glucose or galactose. 9-Methyl 4,8-sphingadienine-containing ceramides are usually glycosylated to form fungal cerebrosides, but the recent description of a ceramide dihexoside (CDH) presenting phytosphingosine in Magnaporthe grisea suggests the existence of alternative pathways of ceramide glycosylation in fungal cells. Along with their unique structural characteristics, fungal CMHs have a peculiar subcellular distribution and striking biological properties. In Pseudallescheria boydii, Candida albicans, Cryptococcus neoformans, Aspergillus nidulans, A. fumigatus, and Schizophyllum commune, CMHs are apparently involved in morphological transitions and fungal growth. The elucidation of structural and functional aspects of fungal cerebrosides may therefore contribute to the design of new antifungal agents inhibiting growth and differentiation of pathogenic species.

Structural Chemistry of Polysaccharides from Fungi and Lichens

Publisher Summary This chapter discusses the chemical structures of the polysaccharides of fungi and lichens investigated from 1967 to the middle of 1980. It is convenient to group the polysaccharides in terms of their chemical structures, according to the nature of the component sugars, the predominant linkage and configuration, and, in the case of heteropolymers, the nature of the main chain. The cell walls of Fusicoccum amygdali are stained blue with iodine and attacked by alpha amylase. Extracts of Sporothrix schenckii and Ceratocystis stenoceras contain 4-O-substituted D -glucopyranosyl units and the solutions give a blue color with iodine. Glycogens have been isolated from Candida albicans, Blastocladiella emersonii, Neurospora crassa, Allomyces macrogynus, Rhizophydium sphaerotheca, and Monoblepharella elongata. They have β-amylolysis values of 45–45% and average chain-length (x) values of 11–14 D -glucosyl units. This chapter briefly discusses about glucans including pseudonigeran, pullulan, cellulose etc; mannans including linear mannans etc; galactans; and heteropolysaccharides based on D-mannan main-chains and galactan main-chains.

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy.

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

Monoclonal Antibody to Fungal Glucosylceramide Protects Mice against Lethal Cryptococcus neoformans Infection

ABSTRACT Glucosylceramides (GlcCer) are involved in the regulation of Cryptococcus neoformans virulence. In the present study, we demonstrate that passive immunization with a monoclonal antibody to GlcCer significantly reduces host inflammation and prolongs the survival of mice lethally infected with C. neoformans, revealing a potential therapeutic strategy to control cryptococcosis.

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

ABSTRACT We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Glycolipids from macroalgae: potential biomolecules for marine biotechnology?

Brown, red and green algae from the Southeastern coast of Brazil were successively extracted with chloroform/methanol 2:1 and 1:2 (v/v). The crude lipid extract was partitioned according to Folch and the lower phase enriched in glycolipids was fractionated on a silica gel column chromatography eluted with chloroform, acetone and methanol. Three major orcinol-reactive bands present in the acetone and methanol fractions were detected by thin-layer chromatography with chromatographic mobilities corresponding to sulfoglycolipids and glycosyldiacylglycerols. These fractions exhibited potent antiviral activity against HSV-1-ACVs and HSV-1-ACVr and present low toxicity for cell cultures. Purification and identification of these bioactive glycolipids will be necessary in order to elucidate their primary structures and mechanism of action.

Fungal glycans and the innate immune recognition

Polysaccharides such as α- and β-glucans, chitin, and glycoproteins extensively modified with both N- and O-linked carbohydrates are the major components of fungal surfaces. The fungal cell wall is an excellent target for the action of antifungal agents, since most of its components are absent from mammalian cells. Recognition of these carbohydrate-containing molecules by the innate immune system triggers inflammatory responses and activation of microbicidal mechanisms by leukocytes. This review will discuss the structure of surface fungal glycoconjugates and polysaccharides and their recognition by innate immune receptors.

Structure, Cellular Distribution, Antigenicity, and Biological Functions of Fonsecaea pedrosoi Ceramide Monohexosides

ABSTRACT Monohexosylceramides (CMHs, or cerebrosides) have been reported as membrane and cell wall constituents of both pathogenic and nonpathogenic fungi, presenting remarkable differences in their ceramide moiety compared to mammalian CMHs. Current evidence suggests that CMHs are involved in fungal differentiation and growth and contribute to host immune response. Here we describe a structural diversity between cerebrosides obtained from different forms of the human pathogen Fonsecaea pedrosoi. The major CMH species produced by conidial forms displayed the same structure previously demonstrated by our group for mycelia, an N-2′-hydroxyhexadecanoyl-1-β-d-glucopyranosyl-9-methyl-4,8-sphingadienine. However, the major cerebroside species purified from sclerotic cells carries an additional hydroxyl group, bound to its long-chain base. The structural difference between cerebrosides from mycelial and sclerotic cells was apparently not relevant for their antigenicity, since they were both recognized at similar levels by sera from individuals with chromoblastomycosis and a monoclonal antibody to a conserved cerebroside structure. Preincubation of fungal cells with anti-CMH monoclonal antibodies had no effect on the interaction of F. pedrosoi sclerotic cells with murine macrophages. In contrast to what has been described for other fungal species, sclerotic bodies are resistant to the antifungal action of anti-CMH antibodies. Immunofluorescence analysis showed that recognition of sclerotic cells by these antibodies only occurs at cell wall regions in which melanization is not evident. Accordingly, melanin removal with alkali results in an increased reaction of fungal cells with anti-CMH antibodies. Our results indicate that cerebroside expression in F. pedrosoi cells is associated with dimorphism and melanin assembly on the fungal cell wall.