Análises de glicoesfingolipídeos em diferentes espécies do fungo patogênico do gênero Aspergillus

• Ano de defesa: 2018
• Autor(a) principal: Rodrigues, Karolina Marques [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Aspergillus; Glycosphingolipids; Fungi; Lipids; Glicoesfingolipídeos; Fungos; Lpídeos
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=6326368; https://repositorio.unifesp.br/handle/11600/53060

incidence of serious fungal infections has increased in the last decade, mainly due to the increase in the number of immunocompromised patients, representing a serious cause of morbidity and mortality in this population. Recent works show that fungi are vulnerable to inhibitors of sphingolipid biosynthesis, which led us to study the structures of fungal glycosphingolipids, in order to obtain relevant information on the biosynthesis and functional roles of these molecules and to propose new potential targets for antifungal treatment. Glycosylinositol phosphorilceramides (GIPCs), inositolphosphoryl ceramides (IPCs), and monohexosyl ceramides (CMHs) from A. fumigatus, A. flavus, A. niger and A. nidulans, filamentous fungi causing aspergillosis, were extracted, purified and characterized using methods chromatography, biochemistry and mass spectrometry. By mass spectrometry using electrospray ionization (EISMS) in positive mode, two major peaks corresponding to CMHs of 754 m/z and 756 m/z were identified for all species analyzed. By collisioninduced dissociation (CID), m/z fragments of m/z: 736, 574, 556 and 276 were identified for CMH 754 m/z. Similarly for CMH m/z 756 fragments of m/z 738, 576, 558 and 276. The fragmentation pattern for both CMHs corresponds respectively to [M+H1H2O]+, [M+H1H2OHex]+, [M+H2H2OHex]+, [M+H2H2Oacyl]+, the fragments associated with ceramidecontaining CMH, with structures not described in mammalian CMH, d19:2/h18:1 and d19:2/18:0, respectively. Glucosylceramide and galactosylceramide were characterized by high resolution thin layer chromatography (HPTLC). It was possible to identify 2 different species of IPCs [M+H]+, 926 m/z, and 954 m/z, presenting 282 m/z and 310 m/z fragments for sphingosine t18:0 and t20:0, respectively. The presence of inositol was confirmed using 241 m/z precursor ion mass spectrometry, referring to the inositol phosphate group of these structures. For the characterization of the GIPCs in the different Aspergillus species, we used collision induced dissociation (DIC) in tandem MS with eletrotron ionization (ESI), in positive mode [M+H]+. In this way we were able to detect the 1088 m/z and 1116 m/z ions corresponding to GIPCs with 1 hexose; 1250 m/z and 1278 m/z, GIPCs with 2 hexoses; 1412 m/z and 1440 m/z, corresponding to GIPCs containing 3 hexoses. The GIPCs present A. fumigatus, A. flavus and A. niger were derived from the IPC 926 m/z (containing ceramide 666 m/z) and the IPC 954 m/z (containing ceramid 694 m/z), on the other hand, the A. nidulans GIPCs are derived only from the IPC 926 m/z. The presence of galactofuranose residues in GIPCs was confirmed using the monoclonal antibody MEST1 which recognizes terminal residues of galactofuranose. By immunostaining the HPTLC plates with MEST1 the A. fumigatus, A. flavus and A. niger GIPCs, which had a component recognized by mAb MEST1, have the same chromatographic migration as GIPC called Pb1 of Paracoccidioides brasiliensis. As for A. nidulans, only small reactivity with mAb MEST1 was detected. These results indicate that in the four species studied, both CMHs, IPCs and GIPCs have structures not expressed in mammals, indicating that these molecules as well as the specific enzymes involved in the metabolism of these sphingolipids can be considered as targets for antifungal treatment .