Resumo

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. Although the disease process is well characterized, the fungal expression of genes in vivo is poorly explored. During disease, P. brasiliensis must adapt to a range of environments and survival in any one niche should require the differential expression of genes. With the purpose of identifying antigenic proteins potentially expressed during the fungal infectious process, here we applied the in vivo-induced antigen technology (IVIAT). IVIAT has been used to identify genes expressed during human infection by several microorganisms. A wide array of genes involved in nutrient acquisition, melanin synthesis, adhesion, stress response, general metabolism were induced and have been identified. A P. brasiliensis cDNA expression library was screened in order to identify clones reactive with sera of PCM patients. Specifically, we hypothesized that by using the IVIAT immunological screening, we could identify proteins that play a role during fungal infection.  A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic L-amino acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3.  The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the vivo-induced antigen technology (IVIAT) we identified immunogenic proteins expressed at high levels during infection.  Quantitative real - time RT-PCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis.