Caracterização funcional da proteína Triose fosfato isomerase de Paracoccidioides brasiliensis como potencial adesina
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2008-09-04
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Universidade Federal de Goiás
Resumo
Paracoccidioides brasiliensis, an important human pathogen causative of
paracoccidioidomycosis (PCM), a systemic mycosis with broad distribution in Latin America.
Adhesion to and invasion of host cells are essential steps involved in the infection and
dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to
host extracellular matrix components to establish infection. An adhesin of P. brasiliensiswas
isolated from two dimensional electrophoresis and characterized. Peptides obtained by partial
sequencing of the isolated protein, which presenteda molecular mass of 29 kDa and pI 5.8,
were subjected to sequence analysis of their amino acids, that revealed strong homology to
triose phosphate isomerase (TPI) from several sources. The complete cDNA and gene
encoding TPI of P. brasiliensis (PbTPI) were characterized and both contained an open
reading frame predicted to encode a 249 amino acid protein that presented all the peptides
characterized in the native PbTPI. The complete coding PbTPI cDNA was cloned and over
expressed in Escherichia coli host. The purified recombinant TPI was used to produce
polyclonal antibody in rabbit. By immunoelectron microscopy and Western blot analysis, TPI
was detected in the cell wall and the cytoplasm of the yeast phase of P. brasiliensis. The
expression of PbTPI was analyzed in transition from mycelia to yeast phase. The native
PbTPI is preferentially expressed in the yeast parasitic phase of P. brasiliensis. The
recombinant PbTPI was found to bind to laminin and fibronectin in ligand far-Western blot
assays. TPI binds preferentially to laminin, as determined by peptide inhibition assays. Of
special note, the treatment of P. brasiliensisyeast cells with anti-PbTPI polyclonal antibody
and the incubation of pneumocytes and VERO cells with the recombinant protein promoted
inhibition of adherence and internalization of P. brasiliensisto those in vitrocultured cells.
These observations indicate that TPI could be contribute to the adhesion of the microorganism
to host tissues and to the dissemination of infection.
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PEREIRA, Luiz Augusto. Caracterização funcional da proteína Triose fosfato isomerase de Paracoccidioides brasiliensis como potencial adesina. 2008. 79 f. Tese (Doutorado em Medicina Tropical e Saúde Publica) - Universidade Federal de Goiás, Goiânia, 2008.