Ítem
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Paracoccidioides brasiliensis PbP27 gene: Knockdown procedures and functional characterization
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Autores
Torres, Isaura
Hernandez, Orville
Tamayo, Diana
Muñoz, Jose F.
García, Ana M.
Gómez, Beatriz L.
Restrepo, Angela
McEwen, Juan G.
Fecha
2014
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Blackwell Publishing Ltd
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Resumen
Abstract
Paracoccidioides brasiliensis PbP27 gene encodes a protein localized in both the fungal cytoplasm and cell wall. The parasitic infectious form produces this protein preferentially with the gene's expression varying between the fungus phylogenetic species. The biological function of the native p27 has yet to be determined during either growth of the yeast or host infection. Therefore, in this study, through the use of antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated mitotically stable PbP27 mutants (PbP27 aRNA) with the goal to evaluate the role of p27 in the biology and virulence of this fungus. PbP27 expression was reduced 60-75% in mutants, as determined by real-time PCR in correlation with a decrease in p27 expression. No alterations in the growth curve or in the ability to shift from mycelia to yeast or from yeast to mycelia were observed in PbP27 aRNA strains; however, we did observe a reduction in cell vitality. Moreover, a decrease in cell viability of PbP27 aRNA yeast cells after interaction with IFN-?-stimulated macrophages was detected. Based on these results, we propose that p27 plays a role in yeast cell architecture and represents one of the mechanisms employed by this fungus for its interaction with the monocyte/macrophage system. p27 plays a role in Paracoccidioides brasiliensis yeast cell architecture and represents one of the mechanisms employed by the fungus for its interaction with the monocyte/macrophage system. © 2013 Federation of European Microbiological Societies. Published by John Wiley and Sons Ltd. All rights reserved.
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Keywords
Complementary RNA , Fungal , HLA DR antigen , Oligonucleotide , Protein p27 , Epitope , Fungal protein , Gamma interferon , HLA DR antigen , Peptide , Protein binding , Agrobacterium tumefaciens , Allele , Amino acid sequence , Article , Cell structure , Cell viability , Fungal gene , Fungal virulence , Fungus growth , Gene expression , Gene frequency , Macrophage , Mycelium , Nonhuman , Nucleotide sequence , Paracoccidioides brasiliensis , Pbp27 gene , Protein function , Real time polymerase chain reaction , T lymphocyte , Yeast cell , Chemistry , Drug effects , Epitope mapping , Gene expression regulation , Gene silencing , Genetics , Immunology , Metabolism , Microbiology , Paracoccidioides , Agrobacterium tumefaciens , Fungi , Paracoccidioides brasiliensis , Amino Acid Sequence , Epitope Mapping , Epitopes , Fungal Proteins , Gene Expression Regulation , Gene Knockdown Techniques , HLA-DR Antigens , Interferon-gamma , Macrophages , Paracoccidioides , Peptides , Protein Binding , Antisense RNA , Fungal survival , Gene silencing , P27 , Paracoccidioides brasiliensis , Pbp27
