|MASEY, CHRISTOPHER - University Of Central Florida|
|TETER, KEN - University Of Central Florida|
Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2009
Publication Date: 6/17/2011
Citation: Masey, C.S., Quinones, B., Teter, K. 2011. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis. Methods in Molecular Biology. 739(1):49-59.
Interpretive Summary: Shiga toxins (Stxs) are produced by enterohemorrhagic Escherichia coli, a food- and water-borne pathogen that in the United States alone causes an estimated 73,000 cases of illness per year. These toxins remove a single adenine residue from the 28S rRNA of the eukaryotic 60S ribosomal subunit. The resulting inhibition of protein synthesis generates a cytotoxic effect. Several methods can be used to detect Stx activity against mammalian cells, but there are disadvantages to each technique. A common procedure measures the viability of intoxicated cells by dye exclusion, MTT assay, or similar protocols. This approach requires several days of incubation and often produces poor quantitative data. Another more quantitative method uses the incorporation of radiolabeled amino acids into newly synthesized proteins to measure the Stx-induced inhibition of protein synthesis. This assay requires the handling of radioactive material, is laborious, and can only process a limited number of samples. A quantitative luciferase-based assay has been described that is similar to the system reported here, but the luciferase assay requires several preparatory and processing steps to enact the detection method. Nonradoactive cell-free assays have also been developed, yet these assays require high concentrations of toxin and/or commercially purchased kits to detect the Stx-induced inhibition of protein synthesis. As an alternative to existing technologies, we developed a cell-based assay for the detection of Stx. A Vero cell line with constitutive expression of a destabilized variant (t1/2 = 2 hrs) of the enhanced green fluorescent protein (d2EGFP) is incubated overnight with purified toxin or cell-free culture supernatants from Stx-producing bacteria. Toxin-treated cells degrade d2EGFP and do not produce more of the protein due to the toxin-induced block of protein synthesis. The fluorescent signal from Vero-d2EGFP cells is accordingly lost in proportion to the applied dose of toxin. This simple, quantitative assay provides reproducible data with minimal hands-on effort. The procedure does not require radioisotopes, commercial kits, or additional processing steps. A fluorescent plate reader is required for sample reading, but the only major recurring cost is the use of black 96-well microplates with clear bottoms. As described below, the Vero-d2EGFP assay can also be adapted to screen for toxin inhibitors or to detect other toxins that inhibit protein synthesis.
Technical Abstract: Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to detect other plant and bacterial toxins that arrest protein synthesis.