Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis

Abstract

The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM). It is protective against murine PCM and is a putative virulence factor. The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp. Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points. In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P. brasiliensis isolates after sequencing two PCR products from each fungal sample. The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp. Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein. The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms. The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots. The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine.

FIG. 1

Maximum-likelihood phylogenetic tree resulting from the analysis of 30 gp43 sequences (1,146 bp, encoding the processed antigen) from 17 different P. brasiliensis isolates. The sequences are indicated by the isolate code names (Pb1 to Pb17; Table 1), and clone 2 variants are in italics. Both informative and noninformative sites were considered.

FIG. 2

Southern blot analyses with labeled probes of the gp43 (A and B) and proteinase Lon (C and D) genes. Genomic DNA was restricted with EcoRI (A and C) or BglII (B and D). Lanes: a, Pb1; b, Pb7; c, Pb2; d, Pb5; e, Pb14; f, Pb13; g, Pb12; h, Pb9; i, Pb4; j, Pb10; l, Pb3; m, Pb11; n, Pb6; and o, Pb8. Digestion with EcoRI was partial in lane f. In a second experiment with the LON probe and BglII-cut DNAs of Pb1 to Pb17, it was confirmed that the labeled fragment migrated at 6.5 kb for all isolates except isolates Pb2, Pb3, and Pb4 (data not shown).