Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.

No abstract

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional signi¢cance in Aspergillus species is unknown. We here identi¢ed and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3P P,4P P-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a signi¢cantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, ␤-1,3-glucan content decreased from 2.7-to 7.8-fold, whereas chitin content increased from 4.0-to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the ␤-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

No abstract

Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate. These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.

Heparin is an intracellular product of vertebrate mast cell currently used as exogenous anticoagulant. Despite of the potent biological activities of exogenous heparin, its physiological function has not been clearly established yet. Here, a heparin with similar structure and anticoagulant properties to the mammalian counterpart was shown to occur as the intracellular product of test cells, a cell monolayer that surrounds egg of the invertebrate Styela plicata (Chordata-Tunicata). As in the case of mammalian mast cells, heparin from the ascidian test cells is removed from the intracellular granules after incubation with compound 48/80. Following fertilization, the test cells surrounding the developing larva still retain heparin as metachromatic granulation. In the adult invertebrate, heparin occurs as intracellular granules at the apical tip of epithelial cells surrounding the lumen of both intestine and pharynx, in close contact with the external environment. This is the first description of the presence of heparin in cytoplasmic granules of epithelial-like cells around the lumen of sites exposed to external agents. This arrangement may reflect the participation of heparin in defense mechanisms in this invertebrate.

Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GlcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of β-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galfβ 6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of β-D-galactofuranose. Further studies also showed that inhibition of GlcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GlcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GlcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.