title: A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis.
abstract: Inhibition of protein synthesis is a common mechanism by which bacterial and plant toxins injure human cells. Examples of toxins that inhibit protein synthesis include shiga toxins of Escherichia coli, diphtheria toxin, Pseudomonas exotoxin A and the plant toxin ricin. In order to facilitate studies on toxin pathogenesis and to enable screening for inhibitors of toxin action, a quantitative and highly sensitive assay for the action of these toxins on mammalian cells was developed. The cDNA encoding destabilized luciferase was cloned into an adenoviral expression plasmid and a high-titre viral stock was prepared. Following transduction of Vero cells, luciferase expression was found to be linear with respect to viral multiplicity of infection. Luciferase expression by as few as 10 cells was readily detected. Treatment of transduced cells with either cycloheximide or shiga toxin resulted in a decrease in luciferase activity, with a half-life ranging from 1 to 2 h. Inhibition of luciferase expression was evident at toxin concentrations as low as 1 pg ml(-1). The assay was adapted for use in 24-, 96- and 384-well plates, enabling rapid processing of large numbers of samples. Using this approach, susceptibility of Vero, Hep2, Chang, A549, COS-1 and HeLa cells to three different toxins was determined. These results demonstrate that the luciferase-based assay is applicable to the study of numerous cell types, is quantitative, highly sensitive and reproducible. These features will facilitate studies on pathophysiology of toxin-mediated diseases and allow high-throughput screening for inhibitors of cytotoxicity.
authors.affiliation: Departments of Pediatrics and Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St Louis, MO 63110, USA.
authors.forename: Luyi
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Departments of Pediatrics and Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St Louis, MO 63110, USA.
authors.forename: David B
authors.initials: DB
authors.lastname: Haslam
mesh_terms: D000256:Adenoviridae; D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis* / Q000633:toxicity*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003513:Cycloheximide / Q000633:toxicity; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008163:Luminescent Measurements / Q000379:methods*; D014176:Protein Biosynthesis; D022621:Shiga Toxin / Q000633:toxicity; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D022621:Shiga Toxin; D003513:Cycloheximide; D008156:Luciferases