Purification and some properties of Shiga-like toxin from Escherichia coli O157:H7 that is immunologically identical to Shiga toxin.
A cytotoxin to Vero cells (Shiga-like toxin), which was neutralized by antibody against purified Shiga toxin produced by Shigella dysenteriae 1, was purified from Escherichia coli O157:H7, isolated from a patient with hemorrhagic colitis. The purification procedure consisted of ammonium sulfate fractionation, DEAE-cellulose column chromatography, chromatofocusing column chromatography and high performance liquid chromatography. About 200 micrograms of purified Shiga-like toxin was obtained from cell extracts of 14 liters of culture with a yield of about 15%. The purified Shiga-like toxin showed identical physicochemical, biological and immunological properties to those of Shiga toxin. Purified Shiga-like toxin and Shiga toxin also had the same mobilities on polyacrylamide disc gel electrophoresis and polyacrylamide gel isoelectrofocusing. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, purified Shiga-like toxin migrated as two bands corresponding to the A and B subunits, and these migrated to the same positions as A and B subunits of Shiga toxin. The amino acid composition of the purified Shiga-like toxin was also similar to that of Shiga toxin. The purified Shiga-like toxin showed various biological activities: lethal toxicity to mice when injected intraperitoneally, the LD50 being 30 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 6 pg; and fluid accumulation in rabbit ileal loops at concentrations of more than 1.25 micrograms/loop. These values are comparable with those obtained with Shiga toxin. In an Ouchterlony double gel diffusion test, the lines formed by the purified Shiga-like toxin and Shiga toxin fused, indicating that the two toxins were immunologically identical.
Noda
M
M
Department of Bacterial Infection, Institute of Medical Science, University of Tokyo, Japan.
Yutsudo
T
T
Nakabayashi
N
N
Hirayama
T
T
Takeda
Y
Y
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001427:Bacterial Toxins / Q000276:immunology* / Q000302:isolation & purification / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003093:Colitis, Ulcerative / Q000382:microbiology; D004926:Escherichia coli / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D051379:Mice; D008815:Mice, Inbred Strains; D022622:Shiga Toxin 1; D022603:Shiga Toxins; D012762:Shigella dysenteriae / Q000737:chemistry*; D014709:Vero Cells
D000596:Amino Acids; D001427:Bacterial Toxins; D022622:Shiga Toxin 1; D022603:Shiga Toxins