RESUMO
Acidic glycosphingolipid components were extracted from the mycelium form of the thermally dimorphic mycopathogen Sporothrix schenckii. Two fractions from the mycelium form (Ss-M1 and Ss-M2), having the highest Rf values on HPTLC analysis, were isolated and their structures elucidated by 1- and 2-D 13C- and 1H-nuclear magnetic resonance spectroscopy, and electrospray ionization mass spectrometry with lithium adduction of molecular ions. The structures of Ss-M1 and Ss-M2 were determined to be Manalpha1-->Ins1-P-1Cer and Manalpha1--> 3Manalpha1-->Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester). The Manalpha1-->6Ins motif is found normally in diacylglycerol-based glycophosphatidylinositols of Mycobacteria, but this is the first unambiguous identification of the same linkage making up the core structure of fungal glycosylinositol phosphorylceramides (GIPCs). These results are discussed in relation to the structures of GIPCs of other mycopathogens, including Histoplasma capsulatum and Paracoccidioides brasiliensis.
Assuntos
Ceramidas/química , Esfingolipídeos/química , Sporothrix/química , Agaricus/química , Isótopos de Carbono , Ceramidas/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Molecular , Espectrometria de Massas por Ionização por Electrospray , Esfingolipídeos/isolamento & purificaçãoRESUMO
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.