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.

Two major acidic glycolipid components (Pb-1 and Pb-2) have been extracted from the mycopathogen Paracoccidioides brasiliensis, a thermally dimorphic fungus endemic to rural areas of South and Central America. Sera of all patients exhibiting paracoccidioidomycosis were found to be reactive with Pb-1, but not with Pb-2; no reactivity was observed with sera of healthy patients or those with histoplasmosis [Toledo, M. S., Suzuki, S., Straus, A. H., and Takahashi, H. K. (1995) J. Med. Vet. Mycol. 33, 247-251]. We report here the complete structure elucidation of both P. brasiliensis glycolipids using monosaccharide, fatty acid, sphingosine, and inositol component analysis by GC-MS; 1H- and 31P NMR spectroscopy; ESI-MS and -MS/CID-MS, linkage analysis, and exoglycosidase digestion. The compounds were found to be glycosylinositol phosphorylceramides (GIPCs) with the following structures: Pb-2, Manpalpha1-->3Manpalpha1-->2Ins1-P-1Cer; and Pb-1, Manpalpha1-->3[Galfbeta1-->6]Manpalpha1-->2Ins1- P-1Cer. The serologically nonreactive Pb-2 appears to be a biosynthetic intermediate between mannosylinositol phosphorylceramide (MIPC), which is widely distributed among fungi, and the antigenic Pb-1. Pb-1 is a novel glycosphingolipid, similar to a triglycosyl IPC (Hc-VI) reported from Histoplasma capsulatum [Barr, K., Laine, R.A, and Lester, R. L. (1984) Biochemistry 23, 5589-5596], but differing in the anomeric configuration of the terminal Galf1-->6 residue, which is immunodominant. The significance of these structures as serological and taxonomic markers, as well as their potential utility as targets for immunodiagnostic agents, is discussed.

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