Partial purification and characterization of a cytotoxin from Clostridium difficile

Abstract

A trypsin-sensitive, heat-labile cytotoxin was purified from the supernatant of a culture of Clostridium difficile by a procedure that included ultrafiltration, precipitation with (NH4)2SO4, gel filtration, and ion-exchange chromatography. The procedure resulted in recovery of 20% of the cytotoxin and an estimated 1,500-fold increase in cytotoxic activity. The minimal amount of protein required to give an actinomorphic response in WI-38 cell cultures was 1.4 ng/ml. The estimated molecular weight of the cytotoxin is 240,000. A cytotoxin having similar properties was purified from the stool of a patient with antibiotic-associated pseudomembranous colitis by (NH4)2SO4 precipitation and gel filtration chromatography. This procedure resulted in a recovery of 26% of the cytotoxin, a 50-fold increase in cytotoxic activity, and a cytotoxic response with a minimum of 12.1 ng of protein/ml.