Characterization of sequences coding for the antigen glyceraldehyde- 3-phosphate dehydrogenase of human pathogenic fungus Paracoccidioides brasiliensis

Abstract

A cDNA (Pbgapdh1, GenBank AY061958) and a partial genomic sequence (AF396657), coding for a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with molecular mass of 36 kDa, of Paracoccidioides brasiliensis, isolate Pb01 (ATCC MYA 826), were cloned and characterized. The genomic fragment was obtained by PCR amplification of P. brasiliensis DNA using degenerated primers designed based on the amino acid sequence of the GAPDH of this pathogen. The fragment obtained, with 1253 pb, presenting identity match with GAPDHs, was used in the screening of a cDNA library of P. brasiliensis in the 74 yeast phase. The cDNA sequence with 1,717 nucleotides presents an open reading frame (ORF) with 1014 pb, coding for a protein (GAPDH1) with a predicted molecular mass of 36 kDa and containing the peptides previously sequenced. The primary structure of PbGAPDH1 was confirmed by comparative analysis to the sequences deposited in data banks. The 1253 bp partial genomic sequence presents four introns interspersed with five exons. The expression of Pbgapdh1 was analyzed by two-dimensional gel electrophoresis of proteins and Northern blot. We detected a single species of mRNA of 1.8 Kb, preferentially expressed in the yeast parasitic phase of P. brasiliensis, in agreement with the high levels of the GAPDH expression in the yeast cells of this pathogen.