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PubMed1988

Secretion of the 43 kDa glycoprotein antigen by Paracoccidioides brasiliensis.

Yeast forms of the pathogenic fungus Paracoccidioides brasiliensis secrete into the culture supernatant a 43,000 daltons glycoprotein (Gp43) which can be immunoprecipitated specifically by sera from patients with paracoccidioidomycosis. We show here that following labelling of P. brasiliensis with (35S)methionine, Gp43 was detected as the major component in the culture supernatant fluid as early as 1 hour after addition of the radiolabel. The amount of Gp43, as determined by a competitive radioimmunoassay, or by staining total protein after sodium dodecylsulfate-polyacrylamide gel electrophoresis, progressively increased in the culture supernatant until the culture reached the late exponential phase. It then decreased and continued to do so in the stationary phase. These results indicate that Gp43 is continuously produced and secreted in the medium by actively growing yeasts and that cultures in the exponential phase of growth should be used for a maximal yield of this exocellular antigen.

Antigens, Fungal↗

PubMed1986

Exocellular components of Paracoccidioides brasiliensis: identification of a specific antigen.

Yeast forms of Paracoccidioides brasiliensis grown in liquid medium produced exocellular components. Immunodiffusion reactions and immunoprecipitations of 131I-radiolabeled antigenic components with sera from patients having paracoccidioidomycosis (PCM) were used to monitor the isolation of specific constituents. Components having the main antigenic activity (fCon A) were isolated by exclusion from a Bio-Gel P30 column, followed by successive binding of eluted material to a Sepharose-concanavalin A column, and elution. The product contained, from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, a minor 43,000-molecular-weight (MW) component (gp43), a polydisperse high-MW glycoconjugate, and a diffusely migrating 55,000-MW glycoprotein (gp55). Other components, including a 72,000-MW glycoprotein, were irregularly expressed. The high-MW glycoconjugate complex contained, on the basis of methylation and 13C nuclear magnetic resonance data, a branched structure of mainly mannopyranosyl units. These were nonreducing ends, 6-O-, 2-O-, and 2,6-di-O-substituted, and the specific rotation of +16 degrees indicated that the glycosidic configurations of the units were alpha and beta in a ratio of ca. 1:1 (concanavalin A binding indicated that nonreducing ends or 2-O-substituted units or both of alpha-D-mannopyranose were present). A small proportion of nonreducing end units of D-galactopyranose were also present in this polysaccharide. gp55 is a glycoprotein containing a complex carbohydrate moiety with fucose, mannose, galactose, and glucose, either as terminal nonreducing units or substituted in positions indicated by methylation data. Both PCM and normal human sera precipitated the high-MW glycoconjugate from 131I-labeled fCon A preparations, whereas gp55 was unreactive with human sera. gp43 was a specific antigenic component of P. brasiliensis culture filtrates which could be isolated in a pure form by gel filtration column chromatography (Sephadex G150) or by Sepharose-patient immunoglobulin G affinity chromatography. 131I-labeled gp43 reacted equally well with 10 PCM sera and hyperimmune rabbit serum against the band E antigen of Yarzabal at a 10(-3) dilution. At the same dilution, no reaction was detected with sera from normal individuals and from patients with other mycoses. Similarly, only PCM sera and the hyperimmune anti-E serum gave precipitin lines with gp43 in the less sensitive immunodiffusion tests. gp43 consisted of three components, with pI 6.7, 6.4 and 6.2, all of which reacted with PCM serum.

Antigens, Fungal↗