A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a ␤-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogoldantibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the ␤-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal ␤-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.

Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.

Pseudallescheria boydii is a fungal pathogen that causes disease in immunocompromised patients. Ceramide monohexosides (CMHs) were purified from lipidic extracts of this fungus, showing that, as described for several other species, P. boydii synthesizes glucosylceramides as major neutral glycosphingolipids. CMHs from P. boydii were analyzed by high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. These combination of techniques allowed the identification of CMHs from P. boydii as molecules containing a glucose residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids. Antibodies from a rabbit infected with P. boydii recognized CMHs from this fungus. Antibodies to CMH were purified from serum and used in indirect immunofluorescence, which revealed that CMHs are detectable on the surface of mycelial and pseudohyphal but not conidial forms of P. boydii, suggesting a differential expression of glucosylceramides according with morphological phase. We also investigated the influence of antibodies to CMH on growth and germ tube formation in P. boydii. Cultures that were supplemented with these antibodies failed to form mycelium, but the latter was not affected once formed. Similar experiments were performed to evaluate whether antibodies to CMH would influence germ tube formation in Candida albicans, a fungal pathogen that synthesizes glucosylceramide and uses differentiation as a virulence factor. Addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. These results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.

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 ␣-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that ␣-glucan of P. boydii is a glycogenlike polysaccharide consisting of linear 4-linked ␣-D-Glcp residues substituted at position 6 with ␣-D-Glcp branches. Soluble ␣-glucan, but not ␤-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when ␣-glucan from conidial surface was removed by enzymatic treatment with ␣-amyloglucosidase, thus indicating an essential role of ␣-glucan in P. boydii internalization by macrophages. ␣-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with ␣-amyloglucosidase. Finally, ␣-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 ␣-glucan could be a target for intervention during fungal infections.

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.