Resumo

Sporotrichosis is a chronic subcutaneous mycosis that affects both humans and animals worldwide. It is caused by the traumatic inoculation of fungus Sporothrix schenckii, recently cleared as component of a complex related to several cryptic infectious species. One of the most thoroughly studied antigenic component of S. schenckii complex is a 70-kDa glycoprotein (Gp70), present in the walls of both fungal forms (mycelium and yeast), which can mediate fungus adhesion to host tissues and to matrix proteins of the basal lamina. The Gp70 becomes a target that might be exploited in the development of new chemotherapeutic modalities against sporotrichosis. In light of this, the aim of this study was to compare different methods for protein extraction from the yeast phase of S. schenckii ATCC 16345. Proteins were extracted using three different protocols: collecting of antigens released naturally (exoantigen) by cultures in YCG medium, extraction of cell wall and whole-cell proteins using, respectively, glass beads and yeast protein extraction reagent (Y-PER). Proteins samples were purified by precipitation with 70% ethanol at 4ºC for 18 h and then submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After denaturing electrophoresis in 10% gels, protein bands were stained with Coomassie-blue and analyzed. Despite protein profiles having revealed differences between extraction methods, the characteristic S. schenckii Gp70 was identified in all samples. Of all three methods, Y-PER led to gels with less artifacts and better defined bands. This method could be indicated for efficient extraction and characterization of 70 kDa glycoprotein and to aid in the development of an efficient therapy for sporotrichosis. Financial Support: PAEDEX /AUIP/PROPG