Characterization of the RNA and carbohydrate content transported in fungal extracellular vesicles

• Grant number: 10/19410-6
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: February 01, 2011
• End date: December 31, 2014
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Rosana Puccia
• Grantee: Roberta Peres da Silva
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated scholarship(s): 13/22200-1 - Terminal oligosaccharide profile of Paracoccidioides extracellular vesicle surface, BE.EP.DR

Abstract

We have shown that Paracoccidioides brasiliensis in yeast phase can carry multiple proteins to the extracellular medium through membranous/exosome vesicles. These proteins have different functions, but potentially interact. Sera from patients with paracoccidioicomicose recognize several vesicular components, including epitopes containing residues of highly immunogenic a-galactosyl found not only in in vesicles, but also in cell wall and intracellular vacuoles. Vesicle preparations were able to stimulate macrophages in vitro and their cytokine production profiles were opposite in Pb3 (less virulent; PS2 group) when compared with Pb18 (virulent group S1). This project aims to continue the work with a functional approach and comparison between Pb3 and Pb18, highlighting four main objectives: 1) to fractionate (membrane and soluble fraction; lipid and protein fractions) extracellular vesicles Pb3 and Pb18 and verify the location of stimulating components of macrophages in vitro. In parallel, we will evaluate the presence of genetic material, polysaccharide and peptides in vesicular preparations; 2) to verify the in vivo effect of Pb3 and Pb18 vesicle preparations before and after challenge with these isolates; 3) to verify the presence of extracellular vesicles in culture supernatants from the mycelial phase. If the result is positive, to characterize the protein content and the presence of epitope-Gal in the vesicles; 4) to express and study selected proteins after proteome analysis of vesicles from Pb18 and Pb3. As vesicles are recovered in modest amounts from the culture supernatant, the viability of some objectives can be evaluated only during the development of the project. The project is related to the thematic project coordinated by the supervisor (06-05095-6) which is expected to be concluded in November 2011. (AU)