ÿþ<HTML><HEAD><TITLE>XI International Meeting on Paracoccidioidomycosis</TITLE><link rel=STYLESHEET type=text/css href=css.css></HEAD><BODY aLink=#ff0000 bgColor=#FFFFFF leftMargin=0 link=#000000 text=#000000 topMargin=0 vLink=#000000 marginheight=0 marginwidth=0><table align=center width=700 cellpadding=0 cellspacing=0><tr><td align=left bgcolor=#cccccc valign=top width=550><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=3><font size=1>XI International Meeting on Paracoccidioidomycosis</font></font></strong><font face=Verdana size=1><b><br></b></font><font face=Verdana, Arial,Helvetica, sans-serif size=1><strong> </strong></font></font></td><td align=right bgcolor=#cccccc valign=top width=150><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=1><font size=1>Resume:30-1</font></em></font></strong></font></td></tr><tr><td colspan=2><br><br><table align=center width=700><tr><td><b>Investigação</b><br><table width="100%"><tr><td width="60">30-1</td><td><b>Does Paracoccidioides brasiliensis use extracellular vesicles to communicate with the host?</b></td></tr><tr><td valign=top>Authors:</td><td><u>Rosana Puccia </u> (UNIFESP-EPM - Universidade Federal de São Paulo) </td></tr></table><p align=justify><b><font size=2>Abstract</font></b><p align=justify class=tres><font size=2>We have characterized extracellular vesicles from Paracoccidioides brasiliensis. We used isolate Pb18 (main species S1) as a model, in comparison with Pb3 (phylogenetic group PS2). We showed that P. brasiliensis antigens were present in vesicle preparations from both isolates, as observed in immunoblots revealed with sera from PCM patients. We demonstrated that vesicles carry highly immunogenic &#945;-linked galactopyranosyl (&#945;-Gal) epitopes. TEM images revealed immunogold labeling with Marasmius oreades agglutinin (MOA), which recognizes terminal &#945;-Gal, on the surface and in the lumen of Pb18 vesicles; anti-&#945;-Gal abundantly labeled the cell wall and inside vacuoles. We characterized the secretome of Pb18 using tandem mass spectrometry. We found 123 proteins highly enriched or exclusively identified in vesicles, against 200 in vesicle-free fractions and 105 detected in both fractions at different percentages of abundance relative to total secretome. Vesicle proteins showed diverse functions, but groups containing signaling proteins, GTPase-mediated signal transduction, cell division, and nucleosome organization were highly enriched in vesicles relative to whole genome. Interacting network detected 45 Pb18 vesicles proteins with enriched biological process GO categories. Vesicle lipids were fractionated in silica-gel 60 and analyzed. In Pb18 vesicles, two different species of phosphatidylinositol, namely alkyl-C16:0-acyl-C18:1 and alkyl-C16:0-acyl-C18:2, were absent in Pb3. The predominant fatty acid in Pb3 vesicles was oleic acid (C18:1) followed by linoleic acid (C18:2), which was the most abundant in Pb18 vesicles. The prevalent sterol in Pb3 and Pb18 vesicles was ergosta-7,22-dien-3&#946;ol, at a ratio of 8 in Pb3 and 1.13 in Pb18. Extracellular vesicles from Pb3 and Pb18 were both able to stimulate cytokine production in RAW264.7 macrophages. However, tumor necrosis factor-&#945; (TNF-&#945;) production was higher when cells were stimulated with vesicles isolated from Pb3, while the production of interleukin-10 was higher when macrophages were stimulated with Pb18 vesicles. Thus, P. brasiliensis vesicles carry immunomodulatory components that might differentially interfere with the host-fungal relationship. Support: FAPESP, CNPq, NIH (grants # 5G12RR008124-16A1 and 5G12RR008124-16A1S1)</font></p><br><b>Keyword: </b>&nbsp;vesicles, secretome, lipidome</td></tr></table></tr></td></table></body></html>