Novel cell-based method to detect Shiga toxin 2 from <i>Escherichia coli </i>O157:H7 and inhibitors of toxin activity

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TitleNovel cell-based method to detect Shiga toxin 2 from Escherichia coli O157:H7 and inhibitors of toxin activity
Publication TypeJournal Article
Year of Publication2009
AuthorsQuiñones, B, Massey, S, Friedman, M, Swimley, MS, Teter, K
JournalAppl Environ MicrobiolAppl Environ MicrobiolAppl Environ Microbiol
Volume75
Pagination1410-6
Date PublishedMar
ISBN Number1098-5336 (Electronic)0099-2240 (Linking)
Accession Number19139230
AbstractEscherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1- and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1- and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Vero-d2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.
Short TitleApplied and environmental microbiologyApplied and environmental microbiology
Alternate JournalApplied and environmental microbiology