Moléculas de microdomínios de membrana de fungos relevantes para a infectividade

• Autor(a) principal: Lacerda Filho, Tanil Gois [UNIFESP]
• Data de Publicação: 2018
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=6433304; https://repositorio.unifesp.br/handle/11600/53039
• topic: Membrane micro-domains; Pathogenic pathogens; Protoplasts; Host-parasite interactions, infectivity; Protoplastos; Interações hospedeiro-parasita; Infectividade; Microdomínios de membrana

Membrane microdomains represent a membrane fraction enriched with glycosphingolipids, sterols and specific glycoproteins and they play a key role in several biological events such as the infectivity of pathogenic fungi. In the past few years we have characterized yeast membrane microdomains of Histoplasma capsulatum, and we have shown that these regions are enriched with specific proteins such as the 32 kDa lamininbinding protein (p32). Also we have shown that the removal of ergosterol from the plasma membrane with methylβcyclodextrin (mβCD) reduces significantly the ability of H. capsulatum to infect alveolar macrophages. The p32 was isolated from membrane fractions resistant to nonionic detergent (Brij 1% at 4ºC) obtained by sucrose gradient ultracentrifugation and by immunoprecipitation with laminin and antilaminin antibodies conjugated to SepharoseproteinA. The immunoprecipitate was submitted to SDSPAGE and the band corresponding to p32 isolated from the gel, submitted to tryptic digestion. The five tryptic fragments were analyzed by mass spectrometry and it was characterized by Mascot software and the database from NCBI. In order to investigate whether other components of fungal membrane microdomains, such as glucosylceramide could also modulate infectivity, we performed experiments using Candida albicans control yeasts and protoplasts obtained after Zymolase treatment (2mg/ml 2 hours) and immunostaining with antiglucosylceramide antibody (MEST2). By confocal microscopy we observed distribution differences of glucosylceramide between protoplasts and control yeasts. In the interaction of C. albicans protoplasts with host cells (1h incubated with macrophages cultures) a polarization of the protoplast glucosylceramide was observed in the region of interaction with the macrophage, not detected in control yeasts. The data presented here may help future studies on the interaction fungi/host cell and eventually help the development of new drugs acting on infections caused by this class of fungi.