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:92-1</font></em></font></strong></font></td></tr><tr><td colspan=2><br><br><table align=center width=700><tr><td><b>Poster (Painel)</b><br><table width="100%"><tr><td width="60">92-1</td><td><b>Silencing of PbGP43 in Paracoccidioides brasiliensis by Antisense RNA technology.
</b></td></tr><tr><td valign=top>Authors:</td><td><u>Isaura Torres Gomez </u>  (U DE A, MEDELLIN, CO - Biology InstituteBCM, CIB - Cellular and Molecular Biology Unit, CIB, Medellin, Colombia) ; Orville Hernandez Ruiz   (U DE A, MEDELLIN, CO - Biology InstituteBCM, CIB - Cellular and Molecular Biology Unit, CIB, Medellin, Colombia) ; Diana Tamayo   (U DE A, MEDELLIN, CO - Biology InstituteBCM, CIB - Cellular and Molecular Biology Unit, CIB, Medellin, Colombia) ; Jose F Munoz   (U DE A, MEDELLIN, CO - Biology InstituteBCM, CIB - Cellular and Molecular Biology Unit, CIB, Medellin, Colombia) ; Ana Maria Garcia   (BCM, CIB - Cellular and Molecular Biology Unit, CIB, Medellin, Colombia) ; Agostinho J Almeida   (ICVS - Life and Health Sciences Research Institute, U de Minho) ; Rosana Puccia   (UNIFESP - Department of Microbiology, Immunology and Parasitology) ; Angela Restrepo   (BCM, CIB - Cellular and Molecular Biology Unit, CIB, Medellin, Colombia) ; Juan G Mcewen   (BCM, CIB - Cellular and Molecular Biology Unit, CIB, Medellin, ColombiaU DE A, MEDELLIN, CO - Facultad de Medicina, U de Antioquia, Medellin, Colombia) </td></tr></table><p align=justify><b><font size=2>Abstract</font></b><p align=justify class=tres><font size=2>During the parasitic phase, P. brasiliensis<i>text</i>, produce a complex antigen profile in which, the glycoprotein of 43 kilodaltons (gp43) has been described as the most specific antigen, which is recognized by 100% of sera from patients with either acute or chronic form of Paracoccidioidomycosis (PCM). Gp43 is mostly excreted and present in the cell-free antigen (CFA) in broth and solid culture medium during the yeast cell growth, and has been related with P. brasilienisis virulence during the course of PCM. Although various studies have been addresses to elucidate the role of this protein, this is the first work in which this gene by means of antisense RNA (aRNA) technology and Agrobacterium Tumefaciens<i>text</i>-Mediated Transformation (ATMT), has been silenced. Pb339, a strain with the capacity to secrete high amount of gp43 was used in this study. The higromycin resistance capacity was used to select the transformant isolates and obtained a mitotically stable P. brasiliensis strain (PbGP43-aRNA). The knockdown isolates shows a consistently reduction in gene expression ranging to 75-80%, evaluated by quantitative reversed transcription PCR (RT qPCR) assay performed on the CFX96 system. Antigen production was evaluated by anti-gp43 mouse monoclonal antibody Mab17c and / or with rabbit polyclonal anti-gp43 serum. A decrease production of gp43 exoantigen was observed as demonstrated by western inmunoblot assays. Pb339 (PbWt), PbWt transformed with a construction targeting PbP27 (PbP27-aRNA ) and native purified gp43 were used as control. In summary, aRNA is an applicable silencing mechanism that is viable in P. brasiliensis <i>text</i> a multicellular and multinuclear fungus. Using aRNA technology and ATMT we generated P. brasiliensis<i>text</i> PbGP43-aRNA strain mitotically stable through the time after subsequent subcultures, with a decrease in expression of PbGP43 at transcripcional and translational level as was demostrated. Futher studies will be conducted in order to demostrated some of the functions described for this gene and its behavior in an in vivo model. Acknowledgement: Colciencias proyect 221340820447; CIB and U de Antioquia supported this work. The National Doctoral Program of COLCIENCIAS supported Torres I and Hernández O. We express our gratitude to Departamento de Microbiologia, Imunologia e Parasitologia, disciplina de Biologia Molecular, UNIFESP, SP Brasil for their help and assistance in the immunoblot assays.
</font></p><br><b>Keyword: </b>&nbsp;Paracoccidioides brasiliensis, PbGP43, Gene Silencing, antisense RNA, immunoblot</td></tr></table></tr></td></table></body></html>