Caracterização Molecular e Expressão Heteróloga de um cDNA Codificante para Tiorredoxina do fungo patogênico humano Paracoccidioides brasiliensis
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2006-08-31
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Universidade Federal de Goiás
Resumo
The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is
the etiological agent of Paracoccidioidomycosis (PCM), a human systemic mycosis highly
prevalent in countries of Latin America. P. brasiliensis is subjected to different insults from
human host, such as oxidative stress caused by reactive oxygen species produced by the host
during the infection. Thioredoxin (TRX) is an intracellular redox protein that is required to
maintain redox homeostasis in response to both reductive and oxidative stress conditions in
several organisms. We report here the characterization of a 811 bp cDNA Pbtrx1, encoding a
PbTRX1 of 116 amino acids, with a predicted molecular mass of 12 kDa and pI 5.2. This
putative protein presented one highly conserved active site motif (WCGPC) between TRXs
from several organisms. The phylogenetic analysis performed with PbTRX1 and TRXs from
other organisms, putted P. brasiliensis in the fungi clade. We also performed the prediction of
the secondary structure of PbTRX1 that shows a pattern characteristic of the open twisted
alpha/beta, similar to TRX secondary structures described in other fungus. In order to obtain
the recombinant PbTRX1, the expression construct pGEX-4T-3-trx1 was introduced into
Escherichia coli cells and the expression and purification of the recombinant protein was
obtained. The recPbTRX1 and PbTRX1 from yeast cells extract were found to catalyze the
reduction of insulin. However the PbTRX1 from yeast cells extract treated with H2O2 showed
highly insulin reduction activity than the yeast cells no treated. PbTRX1 was detected by
Western blotting in the extracts from yeast cells growth and from mycelium to yeast
transition. The yeast cells growth was significantly inhibited by H2O2; however the mycelium
to yeast transition was little affected by this oxidant. Semi-quantitative RT-PCR was
employed to analysis the expression of Pbtrx1 gene in response to H2O2. The level of Pbtrx1
transcripts was higher in yeast cells treated with H2O2 than in yeast cells no treated. To realize
how P. brasiliensis deals with oxidative stress is essential to understand the mechanisms
involved in its survival in the host. It may be possible that PbTRX1 enhances survival of P.
brasiliensis in the host, protecting the fungus against the reactive oxygen species and
allowing, in this way, the progress of the infection.
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DOMINGOS, Fernanda de Castro. Cloning expression and insulin reduction activity analysis of a thioredoxin homalogue of human pathologe Paracoccidioides brasiliensis. 2006. 119 f. Dissertação (Mestrado em Ciências Biolóicas) - Universidade Federal de Goiás, Goiânia, 2006.