XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:54-1

Oral / Poster

54-1

IDENTIFYING COMPONENTS OF EXTRACELLULAR VESICLES FROM Paracoccidioides brasiliensis

Authors:

Roberta Peres da Silva (UNIFESP - Universidade Federal de São Paulo) ; Milene Carmes Vallejo (UNIFESP - Universidade Federal de São Paulo) ; Marcio Lourenço Rodrigues (UFRJ - Universidade Federal do Rio de Janeiro) ; Lysangela Ronalte Alves (IBMP - Instituto de Biologia Molecular do Paraná) ; Samuel Goldenberg (IBMP - Instituto de Biologia Molecular do Paraná) ; Rosana Puccia (UNIFESP - Universidade Federal de São Paulo)

Abstract

Accumulating evidence suggests that the release of microbial extracellular vesicles in the host microenvironment contributes to the pathogenesis and modulation of the immune response. In Cryptococcus neoformans, membranous vesicles carry to the external environment capsular glucuronoxilomanana (GXM), which is the major fungal virulence factor. Our group has recently characterized extracellular vesicular structures in the yeast phase of Paracoccidioides brasiliensis. These vesicles carry antigenic components, including α-galoctosyl epitopes, but they can also stimulate macrophages in culture. Vesicle proteomic and lipidomic analysis are being presented in this Meeting. The present work aims at detecting polysaccharides, nucleic acids and peptides in P. brasiliensis vesicles and studying the role of different vesicle components in the interaction with the host. We are also isolating extracellular vesicles from the fungal mycelium phase, however so far our results have been negative. To precipitate yeast phase extracellular vesicles, cell-free supernatants from Pb3 and Pb18 fungal cultures in Hamâ€™s defined medium-glucose were concentrated and ultracentrifuged at 100,000xg. In order to test for the presence of polysaccharides, lipid components were first separated by extraction with methanol:chloroform, and (glyco)proteins were precipitated with trichloroacetic acid. Polysaccharides were detected in the supernatant of both isolates using the phenol-sulfuric method and will be identified in the future. On the other hand, we have been able to identify RNA in extracellular vesicles from Pb18 and Pb3, besides C. neoformans. RNA was isolated using the miRNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturerâ€™s instructions. We found multiple and heterogeneous RNA species in vesicle preparations, whose nature will be investigated by sequencing. It is of note that both mRNA and miRNA have been isolated from human and mouse mast-cell exosomes. They can be transferred to other cells, translated into functional proteins, and are able to modulate translation of several genes, possibly constituting a new form of intercellular communication. Our results will contribute to clarify the structure of extracellular vesicles and the role of their components in the modulation of immune response and host-parasite relationship. Support: FAPESP, CNPq, Capes

Keyword: Paracoccidioides brasiliensis, extracellular vesicles, RNA, polysaccharides

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