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Secretion of the 43 kDa glycoprotein antigen by Paracoccidioides brasiliensis
B U Stambuk1, R Puccia, M L de Almeida
1Departmento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, São Paulo, Brazil.
Abstract:
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.
Insights
The pathogenic fungus Paracoccidioides brasiliensis secretes a glycoprotein (Gp43) during active yeast growth. Maximal yield of this key antigen is achieved using cultures in the exponential growth phase for diagnostic purposes.
Area of Science:
- Mycology
- Immunology
- Biochemistry
Background:
- Paracoccidioides brasiliensis is a fungal pathogen causing paracoccidioidomycosis.
- The yeast form secretes a 43,000 dalton glycoprotein (Gp43).
- Gp43 is specifically immunoprecipitated by sera from infected patients.
Purpose of the Study:
- To investigate the production and secretion kinetics of Gp43 by P. brasiliensis yeast forms.
- To determine optimal culture conditions for maximal Gp43 yield.
Main Methods:
- Radiolabeling of P. brasiliensis with (35S)methionine.
- Detection and quantification of Gp43 in culture supernatant using immunoprecipitation, competitive radioimmunoassay, and SDS-PAGE.
- Analysis of Gp43 levels across different growth phases.
Main Results:
- Gp43 was detected as the major secreted component within 1 hour of radiolabeling.
- Gp43 levels increased progressively in the supernatant during the exponential growth phase.
- Gp43 levels decreased in the stationary phase, indicating continuous production by actively growing yeasts.
Conclusions:
- Gp43 is actively produced and secreted by P. brasiliensis yeasts during growth.
- Cultures in the exponential phase are optimal for maximizing the yield of Gp43.
- Understanding Gp43 secretion kinetics aids in antigen production for diagnostics.