Perfil transcricional e proteômico de Paracoccidioides em resposta à itraconazol e anfotericina B e identificação de compostos com potencial antifúngico
Carregando...
Data
2013-05-02
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Federal de Goiás
Resumo
The thermally dimorphic fungal pathogen Paracoccidioides is the agent of
paracoccidioidomycosis. This disease is characterized by a granulomatous inflammation
with clinical forms ranging from a benign localized infection to a disseminated one. The
triazole drugs are broad-spectrum antifungal agents and are currently used to treat
infections caused by various pathogenic yeast and molds. The mechanism of action of
azoles has been elucidated in some fungi, although little is known in Paracoccidioides.
Here we aim to investigate the mechanism of action of itraconazole on Paracoccidioides
by using Representational Difference Analysis from Paracoccidioides yeast cells grown
in the absence and presence of itraconazole for 1 and 2 h. Among the Paracoccidioides
genes up-regulated by itraconazole were those mainly involved in cellular transport,
metabolism/energy, transcription, cell rescue, defense and virulence. ERG11, ERG6,
ERG3, ERG5 and ERG25 were up-regulated when evaluated in a timely manner. In vivo
infection experiment in mice corroborated in vitro results. The glyoxylate cycle and its
key enzymes isocitrate lyase and malate synthase (MLS) play a crucial role in the
pathogenicity and virulence of various fungi such as the human pathogens. Here, we
describe a study conducted to develop rational ligands as candidates to inhibit receptor
PbMLS. The important step in the search for ligands for this receptor based on structural
homology, molecular docking and molecular dynamics involving scanning virtual
(virtual screening) through the program AutoDock Vina. Identified from the database of
natural compounds (ZINC data bank) potential candidate ligands to inhibit the activity of
PbMLS when compared to the original binder. This process led us to monoterpene indole
alkaloids of the genus Palicourea (Rubiaceae) comprises about 230 species from shrubs
and small trees distributed mainly in tropical regions. From the molecular docking fifteen
compounds were tested as to its effectiveness in inhibiting the activity of PbMLS. The
specific activity of PbMLS was affected by the compounds. Four indol alkaloids showed
ability to reduce the enzyme activity. Since PbMLS is a linked surface protein that
behaves as an anchorless adhesin, and PbICL is here described as adhesin, we also
investigated if those compounds inhibit the adhesion of the protein to extracellular. Twodimensional
gel electrophoresis we used to investigate the proteins expressed
differentially during treatment with itraconazole and amphotericin B. Gels of three
independent biological replicates were digitalized and the images were analyzed using
the ImageMaster 2D Platinum 6.0 software (GE Healthcare). Spot intensities were
normalized and the statistics analyses were estimated by one-way ANOVA. The spots of
interest were excised, in-gel digested with trypsin, and the peptides were then analyzed
by MS and/or MS/MS and and sequenced. The results obtained here should assist in
understanding the mode of action of drugs in Paracoccidioides, and outline studies
identifying compounds with antifungal activity.
Descrição
Palavras-chave
Citação
SILVA NETO, Benedito Rodrigues da. Perfil transcricional e proteômico de Paracoccidioides em resposta à itraconazol e anfotericina B e identificação de compostos com potencial antifúngico. 2013. 167 f. Tese (Doutorado em Medicina Tropical e Saúde Pública) - Universidade Federal de Goiás, Goiânia, 2013.