The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase acts as an adhesin in Paracoccidioides brasiliensis

Abstract

Paracoccidioides brasiliensis is an important human pathogen that causes paracoccidioidomycosis (PCM), a systemic mycosis with broad distribution in Latin America. The invasion of host cells and the adhesion to them are essential steps involved in the infection and dissemination of this pathogen. Furthermore, the pathogen uses its surface molecules to bind to host extracellular matrix components to establish infection. An antigenic adhesin of P. brasiliensis was isolated after two-dimensional gel electrophoresis of total protein of this fungus and characterized. Endoproteinase Lys-C-digest peptides of the purified protein presented a molecular mass of 36 kDa and pI 6.8 and were subjected to sequence analysis of their amino acids, which revealed strong homology to glyceraldehyde-3-phosphate dehydrogenase (GAPDH: EC 1.2.1.12) of several sources. The complete cDNA and gene encoding PbGAPDH were obtained and both contain an open reading frame (ORF) predicted to encode a 338-amino acid protein that presents all the peptides characterized in the native PbGAPDH. The Pbgapdh gene contains five exons interrupted by four introns. The analyses performed with the deduced PbGAPDH suggest its usefulness in providing phylogenetic relatedness, and also evidence the correlation between the phylogeny provided by the deduced proteins and the 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 with the high levels of GAPDH expression in the yeast cells of this pathogen. The purified recombinant GAPDH was used to produce polyclonal antibody in rabbit. Using immunoelectron microscopy and Western blot analysis, we detected GAPDH in the cell wall and the cytoplasm in the yeast phase of P. brasiliensis. The recombinant GAPDH was able to bind to fibronectin, laminin, and type I collagen. Remarkably, both the treatment of P. brasiliensis with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant GAPDH promoted the inhibition of the adherence and the internalization of P. brasiliensis to the in vitro cultured cells. These observations indicate that GAPDH could possibly contribute to the adhesion of the microorganism to the host tissues as well as to the dissemination of the infection.