Functional Characterization of Histoplasma capsulatum Lipid Rafts

The structural organizational of detergent resistant plasma membrane lipid rafts of yeast forms of Histoplasma capsulatum and the relationship of these lipid raft components with fungal ability to infect mouse alveolar macrophage was analyzed by a series of experiments with methyl-beta-cyclodextrin (mβCD) and soluble ergosterol or cholesterol. It was observed the presence of two types of lipid rafts: i) ergosterol-dependent lipid rafts and ii) glycosphingolipid enriched/dependent lipid rafts. About 40% of ergosterol and 25% of glycosphingolipids (GSLs) of H. capsulatum yeasts are present in membrane microdomain fractions resistant to treatment with 1% Brij 98 at 4°C. Specific proteins were also enriched in these lipid rafts, particularly: Pma1p, a fungal plasma membrane proton ATPase and microdomain protein marker; a (glyco)protein of 30kDa, able to bind to laminin and a 50kDa protein recognized by mAb anti-α5-integrin. Removal of ergosterol of H. capsulatum by mβCD reduced the fungal ability to infect alveolar macrophages by 45% and displaced Pma1p and the 30kDa (glyco)protein from lipid raft fractions to detergent soluble fractions. On the other hand, mβCD treatment did not displace GSLs or α5-integrin-like 50kDa protein from lipid raft fractions to detergent soluble fractions. Reinsertion of ergosterol but not cholesterol into membranes of mβCD-treated yeasts restored their ability to infect alveolar macrophages indicating that sterol specific structural features are required for functional organization of microdomains. Among the several components detected in the detergent insoluble membranes of H. capsulatum it is noteworthy the presence of GM1-binding proteins (approximately 18 and 28 kDa) suggesting that H. capsulatum may express a ganglioside binding protein possibly involved in the fungal-host interaction. Studies to characterize the roles of ergosterol, glycosphingolipids and specific proteins in fungal infectivity, regulation of membrane properties and signaling processes may represent new therapeutic approaches to histoplasmosis.

Supported by FAPESP, CNPq and CAPES.