XI International Meeting on Paracoccidioidomycosis
Resume:16-1


Poster (Painel)
16-1THE PATHOGENIC FUNGUS PARACOCCIDIOIDES BRASILIENSIS EXPORTS EXTRACELLULAR VESICLES CONTAINING HIGHLY IMMUNOGENIC α-GALACTOSYL EPITOPES
Authors:Milene Vallejo (UNIFESP - UNIVERSIDADE FEDERAL DE SÃO PAULO) ; Matsuo Alisson (UNIFESP - UNIVERSIDADE FEDERAL DE SÃO PAULO) ; Luciane Ganiko (UTEP - UNIVERSITY OF TEXAS IN EL PASO) ; Lia Medeiros (UFRJ - UNIVERSIDADE FEDERAL DO RIO DE JANEIRO) ; Kildare Miranda (UFRJ - UNIVERSIDADE FEDERAL DO RIO DE JANEIRO) ; Luiz Severino (UNIFESP - UNIVERSIDADE FEDERAL DE SÃO PAULO) ; Edna Haapalainen (UNIFESP - UNIVERSIDADE FEDERAL DE SÃO PAULO) ; Rita Coimbra (UNIFESP - UNIVERSIDADE FEDERAL DE SÃO PAULO) ; Igor Almeida (UTEP - UNIVERSITY OF TEXAS IN EL PASO) ; Rosana Puccia (UNIFESP - UNIVERSIDADE FEDERAL DE SÃO PAULO)

Abstract

Exosome-like vesicles carrying virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we characterized extracellular vesicles from P. brasiliensis isolates Pb3, which represents phylogenetic group PS2, and Pb18 from the main species S1 and widely used in experimental PCM. For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham’s defined medium-glucose were concentrated and ultracentrifuged at 100,000 g. We show here that P. brasiliensis antigens were present in vesicle preparations from both isolates, as observed in immunoblots revealed with sera from PCM patients. We also demonstrate that cell and vesicles carrying highly immunogenic α-linked galactopyranosyl (α-Gal) epitopes. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing α-Gal epitopes reacted strongly with anti-α-Gal antibodies isolated from both Chagas’ disease (Ch anti-α-Gal) and PCM (PCM anti-α-Gal) patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal α-Gal), but only faintly with natural anti-α-Gal. Reactivity was inhibited after treatment with α-galactosidase. TEM images confirmed the ELISA data and revealed immunogold labeling with MOA lectin on the surfaces and in the lumen of Pb18 vesicles. When Pb18 yeast cells were incubated with Ch anti-α-Gal and analyzed by TEM, immunogold particles were abundantly observed on the cell wall, following a punctuated confocal pattern, and inside vacuoles. To better investigate the localization of α-Gal epitopes, we fractionated Pb3 and Pb18 vesicle preparations into lipid and lipid-free fractions and tested the products by ELISA with anti-α-Gal before and after treatment with α-galactosidase. Lipid-free vesicle fractions reacted with anti-α-Gal in ELISA only when not digested with α-galactosidase, while reactivity with glycoproteins was reduced after β-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and α-galactosyl epitopes.਀匀甀瀀瀀漀爀琀㨀 䘀䄀倀䔀匀倀Ⰰ 䌀一倀焀Ⰰ 一䤀䠀 ⠀最爀愀渀琀猀⌀ 㔀䜀㄀㈀刀刀  㠀㄀㈀㐀ⴀ㄀㘀䄀㄀ 愀渀搀 㔀䜀㄀㈀刀刀  㠀㄀㈀㐀ⴀ㄀㘀䄀㄀匀㄀⤀  ਀㰀⼀昀漀渀琀㸀㰀⼀瀀㸀㰀戀爀㸀㰀戀㸀䬀攀礀眀漀爀搀㨀 㰀⼀戀㸀☀渀戀猀瀀㬀倀䄀刀䄀䌀伀䌀䌀䤀䐀䤀伀䤀䐀䔀匀 䈀刀䄀匀䤀䰀䤀䔀一匀䤀匀Ⰰ 䔀堀吀刀䄀䌀䔀䰀䰀唀䰀䄀刀 嘀䔀匀䤀䌀䰀䔀匀Ⰰ ☀⌀㤀㐀㔀㬀ⴀ䜀䄀䰀䄀䌀吀伀匀夀䰀 䔀倀䤀吀伀倀䔀匀㰀⼀琀搀㸀㰀⼀琀爀㸀㰀⼀琀愀戀氀攀㸀㰀⼀琀爀㸀㰀⼀琀搀㸀㰀⼀琀愀戀氀攀㸀㰀⼀戀漀搀礀㸀㰀⼀栀琀洀氀㸀