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Title:   Author  item
                                            Quinones, Beatrizitem
                                            MASEY, SHANE - UNIV.CENTRAL FLA. ORLANDOitem
                                            Friedman, Mendelitem
                                            Swimley, Michelleitem
                                            TETER, KEN - UNIV.CENTRAL FLA. ORLANDO
  Submitted to: Applied and Environmental Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/1/2009
                                    Publication  Date: 3/2/2009
                                    Citation: Quinones, B., Masey, S., Friedman, M., Swimley, M.S, Teter, K. 2009. A Novel Cell-Based Method to Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity. Applied and Environmental Microbiology. 75(5):1410-1416.Interpretive Summary: Shiga toxin-producing Escherichia coli, with E. coli O157:H7 as the most common serotype, is an enteric pathogen known to cause human gastrointestinal illnesses ranging from bloody diarrhea and hemorrhagic colitis to life-threatening hemolytic-uremic syndrome.  It has been estimated that E. coli O157 causes approximately 73,000 cases of illness per year in the United States from food and waterborne sources.  Shiga toxins (Stx1 and Stx2) are major virulence factors in E. coli O157 pathogenicity.  These toxins inhibit protein synthesis by inactivating the ribosome and are thought to contribute to the development of hemolytic uremic syndrome, a potentially fatal disease for which treatment is currently limited to supportive care.  Toxin inactivation would prevent the development of hemolytic-uremic syndrome, but anti-toxin therapeutics are not currently available.  Detection methods to prevent the distribution of E. coli O157 in foods are thus an important component of food safety programs. 
	The rise in foodborne-related outbreaks of E. coli O157 has heightened the importance of developing better methods to rapidly detect and characterize Stxs from E. coli O157 strains.  Several methods have been developed to examine Stx activity against mammalian cells.  Current assays that measure the viability of intoxicated Vero cells require several days of incubation and often produce poor quantitative data.  Other methods that are more quantitative and sensitive measure the incorporation of radioactive amino acids into newly synthesized proteins.  However, these radioactivity assays are complex and laborious and only allow a limited number of conditions to be examined.  A quantitative luciferase-based assay was recently developed to measure Stx toxicity in a high throughput format, but this system requires several preparatory and processing steps to detect luciferase expression.  
	In the present study, we describe a simple cell-based assay for the detection of Stxs and inhibitors of Stx activity.  A Vero cell line that expresses a destabilized variant (t1/2 = 2 hours) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the Stx-induced inhibition of protein synthesis.  Because toxin-susceptible cells will degrade d2EGFP and will not produce more of the protein, exposure to Stx will eliminate the fluorescent signal from Vero-d2EGFP cells.  This assay was used to quantify the dose-dependent inhibition of protein synthesis in Vero-d2EGFP cells exposed to purified Stx2 or to cell-free culture supernatant from a Stx1/2-producing strain of E. coli O157.  We also demonstrated that EGFP fluorescence was maintained in Stx-treated cells when the intoxication process was disrupted.  To capitalize upon this observation, the Vero-d2EGFP assay was used to screen a panel of natural compounds for anti-Stx activities.  We found that grape seed and grape pomace extracts both provided strong cellular protection against Stx, and we confirmed these results with an independent toxicity assay that monitored the overall level of protein synthesis in toxin-treated cells.
                                Technical Abstract: Escherichia coli O157:H7 is a leading cause of foodborne 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 Stxs and inhibitors of Stx activity.  A Vero cell line harboring a destabilized variant (t1/2 = 2 hours) 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 Stx1/2-producing E. coli O157:H7.  Dose response curves demonstrated that the Stx2-induced inhibition of EGFP fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and established a picogram/ml 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 anti-toxin activities.  Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1/2-containing medium in the presence of either grape seed or grape pomace extract.  The anti-toxin 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 Stx activity and can be further utilized to identify toxin inhibitors.


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