Studies on shiga-like toxin produced by enterohemorrhagic Escherichia coli: purification and characterization of the toxin and development of methods for identifying the toxin.

Abstract

A simple purification method using DEAE cellulose column chromatography and immunoaffinity column chromatography was developed for purifying Shiga-like toxin produced by Escherichia coli O157:H7. About 0.75 mg of purified toxin was obtained from 5 liters of culture (62% recovery). The purified toxin was demonstrated to be immunologically, biologically and structurally indistinguishable from Shiga toxin. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Shiga-like toxin. In the ELISA assay, Shigella dysenteriae type 1, Escherichia coli O157:H7 and some strains of Escherichia coli isolated from traveller's diarrhea were positive. Shiga toxin-resistant Vero cells were isolated by treatment of the cells with nitrosoguanidine. Immunofluorescence studies showed that the mutant Vero cells had lost toxin binding capacity. Samples of S. dysenteriae type 1 and E. coli O157:H7 showed cytotoxicity to the parent cells, but not to the mutant cells. Samples of other organisms showed either no cytotoxicity or cytotoxicity to both cell lines. The results suggested that (1) the presence of a receptor for Shiga-like toxin on Vero cells is essential for expression of cytotoxicity of the toxin, (2) the mutant Vero cells could be used to identify Shiga-like toxin producing organisms.