Variable gp43 secretion by Paracoccidioides brasiliensis clones obtained by two different culture methods

Abstract

The main objectives of this study were to obtain clones of Paracoccidioides brasiliensis by two methods (micromanipulation and plating assay) and to determine if the secretion of the 43-kDa glycoprotein (gp43) is dependent on the clonal culture. The results show that the secretion of gp43 is not dependent on clonal cultures. Clones that originally were secretors of this molecule, after subculturing, lost this characteristic; on the other hand, clones that originally did not secrete gp43 began to secrete gp43 after subculturing.

FIG. 1.

Representative Western blot reactions obtained with crude exoantigens (10 μg) from 30 clones of P. brasiliensis (Pb-18) probed with anti-gp43 monoclonal antibody (10 μg/ml); Goat anti-mouse immunoglobulin G (γ-chain specific) serum conjugated with peroxidase was used as the second antibody. Lanes 1 to 30, clones; S, standard purified gp43. Most of the exoantigens were recognized by the anti-gp43 monoclonal antibody, but four clones did not secrete gp43 and consequently were not recognized (clones 3, 4, 5, and 6 presented no gp43).

FIG. 2.

SDS-PAGE of exoantigens (2 μg) obtained from 10 subcultures of P. brasiliensis (Pb-18) clones 3, 4, and 6 and of exoantigens (2 μg) obtained from 10 subcultures of Pb-339 clone 1. Observe the variability in gp43 expression among the subcultures. In Pb-18 clone 3, exoantigens numbers 1 and 7 secreted great amounts of gp43. In Pb-18 clone 4, exoantigens 3, 7, and 8 secreted large amounts of gp43. In Pb-18 clone 6, exoantigens 2, 3, and 9 secreted large amounts of gp43. In Pb-339 clone 1, gp43 was in abundance in exoantigens 1 and 2 but began to decrease after 3. S, standard purified gp43. Lanes 1 to 10, exoantigens of consecutive subcultures.