Resumo

Paracoccidioides brasiliensis, an important human pathogen causing 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. brasiliensis was isolated from gel after two dimensional electrophoresis and characterized. Endoproteinase Lys-C-digest peptides of the purified protein, which presented a molecular mass of 36 kDa and pI 6.8, were subjected to sequence analysis of their amino acids, that revealed strong homology to glyceraldehyde-3-phosphate dehydrogenase (GAPDH: EC 1.2.1.12) from several sources. The complete cDNA and gene encoding PbGAPDH were obtained and both contained an open reading frame predicted to encode a 338 amino acid protein that presented all the peptides characterized in the native PbGAPDH. The Pbgapdh gene contained 5 exons interrupted by 4 introns. Analysis performed with the deduced PbGAPDH suggested its usefulness in providing phylogenetic relatedness, as well as evidenced the correlation between the phylogeny provided by the deduced proteins and introns positions in the cognate genes. The expression of Pbgapdh was analyzed and a single species of mRNA, of 2.0 Kb, preferentially expressed in the yeast parasitic phase of P. brasiliensis, was detected in agreement to the high levels of the GAPDH expression in the yeast cells of P. brasiliensis. The purified recombinant GAPDH was used to produce policlonal antibody in rabbit. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cell wall and the cytoplasm of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand Far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro cultured cells. These observations indicate that GAPDH could be contribute to the adhesion of the microorganism to host tissues and to the dissemination of infection.