Purification and characterization of alpha-toxin produced by Clostridium novyi type A

Abstract

Our study describes the production, purification, and properties of alpha-toxin from Clostridium novyi type A 19402. The bacterium produced maximal amounts of alpha-toxin when grown at 37 degrees C for 72 h in dialysis flask cultures containing brain heart infusion supplemented with 0.75% Tween 80 and 2% glycerol. The alpha-toxin was purified by precipitation with polyethylene glycol 6000, followed by chromatography on Q-Sepharose, phenyl-agarose, and Mono-Q. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the toxin exhibited a single band with an M(r) of 200,000. The toxin also exhibited a single immunoprecipitin arc by crossed immunoelectrophoresis with antiserum against crude toxin. It was stable when stored at 4 degrees C and also following exposure to buffers with pHs in the range of 4 to 7. The toxin had a minimum lethal dose in mice of 5 to 10 ng, caused rounding of a variety of cells in tissue culture, and was negative in the rabbit ileal loop assay. The cytotoxic activity was inhibited by agents that affect receptor-mediated processes, and the toxin was less active on a CHO mutant cell line that is defective in endosomal acidification. The analysis of the amino acid composition revealed an unusually high proline content. The N-terminal sequence is Met-Leu-Ile-Thr-Arg-Glu-Gln-Leu-Met-Lys.