Alpha-toxin production by Clostridium septicum at different culture conditions

Clostridium septicum is responsible for a number of diseases, the most serious of which are a frequently fatal non-traumatic gas gangrene in man and braxy malignant oedema and blackquarter in farm animals. Immunity to these diseases is mediated mainly by antitoxin raised against C. septicum alpha toxin, which has hemolytic, lethal and necrotizing activities. Clostridium septicum produces lethal antigen only in low titre and in a variable way. In addition, the immunogenicity of native toxic filtrates is weak, which results in poor antibody response in animals. The aim of this work was to determine the best culture conditions for obtention of the protective antigen. To do this, the presence of the alpha toxin was identified in culture filtrates by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under different culture conditions. The strain used was C. septicum 3606. The cultures were performed in anaerobic conditions at 37 degrees C. Two systems were employed: (a) batch (0.5% initial glucose concentration); and (b) batch with partial feeding of the carbon source (0.1% glucose concentration) at controlled pH. The hemolytic activity of supernatants was determined using human erythrocytes, and the final biomass concentration was estimated by dry weight determination. The proteins present in supernatants were concentrated by ultrafiltration and identified in 10% SDS-polyacrylamide gels. With partial feeding of the carbon source the final biomass (2.22 g/L) was three times higher than the amount reached in batch system (0.70 g/L), however, no difference was found in the hemolytic activity (16 HU50/mL) showing no correlations between cell growth and hemolytic activity of the supernatants. Electrophoresis of filtrate cultures obtained with partial feeding of the carbon source allowed us to identify several bands, two of them corresponded to non-active alpha-protoxin (46 kDa) and the active toxin (42 kDa) and the aggregate of the active toxin at the seeding line. On the other hand, the ultrafiltrate from batch system showed less number of bands and the 46 kDa band was much weaker, suggesting a lower toxin production in this system. It becomes important to determine culture conditions and correlate with extracellular protein presence to optimize C. septicum antigen protector production.