Effects of cycloheximide and puromycin on cytotoxic activity of Escherichia coli verocytotoxin (Shiga-like toxin).
Verocytotoxin (VT)-producing Escherichia coli is closely associated with hemorrhagic colitis and hemolytic uremic syndrome. The diagnosis of this infection requires the demonstration of VT activity in fecal filtrates or the isolation of VT-producing E. coli from stools. To improve the sensitivity of the Vero cell assay for detecting VT, we investigated the interaction between this toxin and cycloheximide and puromycin, agents which, like VT and the related Shiga toxin, are protein synthesis inhibitors. Cycloheximide-treated cells were found to be about eightfold more sensitive to VT, this effect being most pronounced when the drug was added before the toxin. In contrast, puromycin treatment had an antagonistic effect in that it decreased the sensitivity of the cells to VT. In assays of VT in fecal filtrates, the addition of cycloheximide (at 4 to 8 micrograms/ml) increased the sensitivity without affecting the specificity of the assays. Likewise, the use of cycloheximide led to an increase in the sensitivity of the serum VT-neutralizing antibody test by a factor of over eightfold.
Petric
M
M
Karmali
M A
MA
Arbus
G S
GS
Roscoe
M
M
Louie
S
S
Cheung
R
R
D000818:Animals; D000992:Antitoxins / Q000032:analysis; D001427:Bacterial Toxins / Q000032:analysis / Q000276:immunology / Q000633:toxicity*; D001681:Biological Assay; D002470:Cell Survival; D003092:Colitis / Q000382:microbiology; D003513:Cycloheximide / Q000494:pharmacology*; D003603:Cytotoxins / Q000032:analysis / Q000276:immunology / Q000633:toxicity*; D004926:Escherichia coli; D004927:Escherichia coli Infections / Q000382:microbiology; D005243:Feces / Q000032:analysis; D006801:Humans; D009500:Neutralization Tests; D011691:Puromycin / Q000494:pharmacology*; D022622:Shiga Toxin 1; D014709:Vero Cells
D000992:Antitoxins; D001427:Bacterial Toxins; D003603:Cytotoxins; D022622:Shiga Toxin 1; D011691:Puromycin; D003513:Cycloheximide