28ª REGEM
Reunião de Genética de Microrganismos
09 a 11 de setembro de 2012 - Rafain Palace Hotel & Convention Center - Foz do Iguaçu - PR
Exoproteome analysis of two phylogenetic lineages of
Paracoccidioides genus PbEPM83 and Pb01
Oliveira, Amanda Rodrigues1; Weber, Simone Schneider1; Bailão, Alexandre Melo1, Parente, Juliana Alves1, Soares,
Célia Maria de Almeida1; Borges, Clayton Luiz1
1-
Laboratório de Biologia Molecular/ ICB II /Universidade Federal de Goiás, Goiânia /Goiás, Brazil.
[email protected]
The Paracoccidioides genus is the causative agent of paracoccidioidomycosis (PCM), which comprises four distinct
phylogenetic lineages (PS1, PS3, S1 and Pb01-like). The clinical isolate Pb01 is phylogenetically distinct from others
strains and has been recently defined as a new specie, Paracoccidioides lutzii, whereas the isolate PbEPM83 is a member
of Species 3 (PS3). In this work, we compared the exoproteome of two isolates of Paracoccidioides genus, PbEPM83 and
Pb01. The protein extracts of each strain were submitted to 2-DE gel electrophoresis in three analytical replicates of three
independent biological samples. The 2-DE gels were analyzed with Image Master 2D Platinum v6.0 (GE Healthcare).
To compare the differences in protein expression between isolates, statistical analyzes was applied under volume ratios of
matched spots. Proteins with significant alteration are under selection and characterization by mass spectrometry. Some
differential spots were manually excised and treated for tryptic digestion. Mass spectra were acquired using a MALDI-QTOF mass spectrometer and data was identified by using Mascot software. Until now, the proteomic analysis revealed an
average of 350 and 355 spots in the PbEPM83 and Pb01 secretomes, respectively. This work allowed the identification
of 146 non-redundant protein/isoforms, which corresponded to 75 different proteins. We identified 27 and 52 proteins/
isoforms preferentially secreted in PbEPM83 and Pb01 secretomes, respectively. In addition were identified 67 proteins
were detected as constitutively expressed in both secretomes. The identified proteins were classified in functional categories
according to the MIPS Functional Catalogue Database. Most of the extracellular identified proteins were classified in
Enery, Metabolism and Cell Rescue, Defense and Virulence, including defense proteins as chaperones (heat shock proteins
and peptidyl-prolyl cis-trans isomerase) that are important part of the cell machinery for correct protein folding and help
to protect cells from stress. Formamidase and superoxide dismutase enzymatic assays have been performed with secreted
and soluble extracts of both isolates. Our results showed that 70% of extracellular identified proteins were predicted as
secretory, mostly using non-conventional secretory pathways, according to Fungal Secretome Database (FSD). Our results
of differential secretome between two phylogenetic species include several proteins of interest that add new information
about the mechanisms of pathogenesis and virulence mechanisms of these isolates. Future functional studies are needed
to evaluate the role of those molecules in pathogenesis/virulence of Paracoccidioides isolates.
Financial Support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e
Projetos (FINEP) and Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG).
MICRO032