RESUMO
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-beta-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.
Assuntos
Antígenos de Fungos/química , Antígenos de Fungos/imunologia , Ascomicetos/imunologia , Cerebrosídeos/química , Cerebrosídeos/imunologia , Animais , Anticorpos Antifúngicos/imunologia , Antígenos de Fungos/metabolismo , Ascomicetos/metabolismo , Cerebrosídeos/metabolismo , Lipídeos/química , Macrófagos/imunologia , Melaninas/metabolismo , CamundongosRESUMO
O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5]3-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.
Assuntos
Aspergillus fumigatus/citologia , Aspergillus fumigatus/imunologia , Parede Celular/química , Galactose/análogos & derivados , Galactose/análise , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Oligossacarídeos/química , Galactose/química , Haptenos/imunologia , Espectroscopia de Ressonância Magnética , Metilação , Estrutura Molecular , Oligossacarídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Álcoois Açúcares/análise , Álcoois Açúcares/químicaRESUMO
A crude polysaccharide obtained from mycelium of Fusarium solani by treatment with 2 per center KOH/2h/100§C and fractionated by gel filtration chromatography yielded three fractions denoted L1,L2 and L3. Chemical analysis of the crude polysaccharide showed the presence od 89,5 per center total carbohydrate, 4 per center protin 14 per center uronic acid, traces of phosphate and hexosamine. Mannose, galactose, glucose and unidentifid pentose, were present in a 27.5:34:34.5:4 molar ratio.