Cloning, Characterization, and Epitope Expression of the Major Diagnostic Antigen of Paracoccidioides brasiliensis

Abstract

The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis. In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced. The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides. The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis. A single transcript of 1.5 kilobase pairs was verified in the yeast phase. The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N-glycosylation site. The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans. However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with the fungal glucanases. Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen.

Antifungal and antitumor models of bioactive protective peptides

“…It elicits an immune response that protects against the intratracheal challenge by virulent P. brasiliensis yeast cells. This molecule has been cloned and sequenced (Cisalpino et al 1996). Apart from B cell epitopes, which are beginning to be identified, the gp43 carries an immunodominant epitope that elicits a predominant IFN-γ -mediated Th-1 response.…”

Diversity in Paracoccidioides brasiliensis . The PbGP43 gene as a genetic marker

“…Diversity within the poly-A site was detected both intra-and inter-isolates, considering that about ten 3 0 RACE generated clones were analyzed for each isolate. Therefore, the PbGP43 transcript is between 1,367 and 1,413-bp long, which correlates well with the 1.5 kb originally found in Northern blot [24]. Full sequencing of the EcoRI 3.8-kb insert revealed that the PbGP43 3 0 neighbor is homologous to the gene encoding importin 11 or Ran binding protein 11 (RanBP11), involved in the nucleuscytoplasmatic transport of molecules.…”

“…PbGP43 was the first gene fully characterized in P. brasiliensis [24]. In Pb339, the gene localized to a genomic EcoRI 3.8 kb cloned fragment, from which a 1,981-pb sequence was initially obtained (GenBank accession number U26160).…”

“…Comparison of data generated by both methods indicated the possible existence of haploid, aneuploid and diploid isolates, being therefore inconclusive. On the other hand, both Southern blots of megarestriction fragments with a PbGP43 gene probe [24] and intra-specific polymorphism in the gene sequence [46] suggested diploidy.…”

“…The present review will focus on the genetic polymorphism observed in the species, which culminated with the detection of phylogenetic species as a result of a multilocus study [23]. Polymorphism in the PbGP43 gene [24] is detailed. This gene encodes fungal glycoprotein gp43, a dominant P. brasiliensis antigen largely studied in the last two decades since its characterization, purification, and production of monoclonal antibodies anti-gp43 [25, 26a, 26b, 27].…”