PMID:19139230

Citation	Quiñones, B, Massey, S, Friedman, M, Swimley, MS and Teter, K (2009) Novel cell-based method to detect Shiga toxin 2 from Escherichia coli O157:H7 and inhibitors of toxin activity. Appl. Environ. Microbiol. 75:1410-6
Abstract	Escherichia 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.
Links	PubMed PMC2648150 Online version:10.1128/AEM.02230-08
Keywords	Animals; Antidotes/pharmacology; Bacteriological Techniques/methods; Cercopithecus aethiops; Escherichia coli O157/pathogenicity; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/biosynthesis; Escherichia coli Proteins/toxicity; Genes, Reporter; Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; Humans; Plant Extracts/pharmacology; Protein Biosynthesis/drug effects; Sensitivity and Specificity; Shiga Toxin 2/antagonists & inhibitors; Shiga Toxin 2/biosynthesis; Shiga Toxin 2/toxicity; Vero Cells
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SHIDY:STXA		GO:0017148: negative regulation of translation	ECO:0000314:		P		Figure 3 shows a drastic decrease in protein synthesis in presence of StxA	complete
ECO57:Q7DI68		GO:0017148: negative regulation of translation	ECO:0000314:		P		Figure 2 shows a decrease level of protein synthesis in presence of shiga toxin 2 (Stx2).	complete CACAO 4385

References

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