title: Use of recombinant gp43 isoforms expressed in Pichia pastoris for diagnosis of paracoccidioidomycosis.
abstract: gp43 is the main diagnostic antigen for paracoccidioidomycosis (PCM). In vitro, gp43 expression in supernatant fluids of Paracoccidioides brasiliensis cultures can be unstable, and its regulation is poorly understood. We have been able to express soluble recombinant gp43 (gp43r) isoforms as N-mannosylated proteins secreted in the supernatants of Pichia pastoris cultures induced with methanol. They were secreted as major components from day 2 of induction and could be purified with affinity columns containing anti-gp43 monoclonal antibodies. We have expressed P. brasiliensis GP43 (PbGP43) sequences from genotypes A, D, and E, and the correspondent gp43r isoforms (gp43r A, -B, and -C, respectively; 200 ng) were compared to native gp43 in immunodiffusion (ID) and dot blot assays. Among 90 PCM patient sera showing ID-positive reactions with purified native gp43, 100% were positive with gp43rD and gp43rE and 98% reacted with gp43rA. Of these sera, 78 were tested in dot blot assays at a 1:1,000 dilution, and 100% reacted with all recombinant isoforms. In ID assays, the specificity was 100%, since 40 sera from patients with related mycoses and 30 sera from healthy individuals did not react with any of the antigens. In dot blot assays, 100% specificity for PCM occurred when cross-reactive mannose epitopes were neutralized with 10 mM metaperiodate or eliminated through deglycosylation. However, a 1:1,000 serum dilution was already discriminatory for most sera. We suggest that P. pastoris recombinant gp43, especially isoforms D and E, may replace the native antigen in ID and dot blot assays for diagnosis and prognosis of PCM. Regulated expression of large amounts of antigen in nonpathogenic yeast would justify its preferred use.
authors.affiliation: Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo, Rua Botucatu 862, oitavo andar, São Paulo, SP 04023-062, Brazil.
authors.forename: K C
authors.initials: KC
authors.lastname: Carvalho
authors.forename: M C
authors.initials: MC
authors.lastname: Vallejo
authors.forename: Z P
authors.initials: ZP
authors.lastname: Camargo
authors.forename: R
authors.initials: R
authors.lastname: Puccia
mesh_terms: D000946:Antigens, Fungal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D010229:Paracoccidioidomycosis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: C103180:43 kDa protein, Paracoccidioides; D000946:Antigens, Fungal; D005656:Fungal Proteins; D006023:Glycoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins