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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: UJNR Food & Agricultural Panel Proceedings
                                    Publication Type: Proceedings
                                    Publication Acceptance Date: 7/31/2008
                                    Publication  Date: N/A
                                    Citation: N/AInterpretive Summary: Shiga toxin-producing Escherichia coli O157:H7 is an enteric pathogen that is known to cause human gastrointestinal illnesses, ranging from bloody diarrhea, hemorrhagic colitis and life-threatening hemolytic-uremic syndrome (HUS).  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 (Stx), 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 (HUS), a potentially fatal disease for which treatment is currently limited to supportive care.  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 Stx from E. coli O157 strains.  Current methods for Stx activity that measure cell viability in Vero cells require several days of incubation and often produce poor quantitative data.  Other assays 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.  Recently, a quantitative luciferase-based assay for measuring Stx cytotoxicity that allows multiple sample analysis was developed.  However, the use of the luciferase system requires several preparatory and processing steps for measuring 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.  This assay was used to quantify the dose-dependent inhibition of protein synthesis in Vero-d2EGFP cells exposed to a Stx1/2-containing E. coli O157 culture supernatant or to purified Stx2.  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.
                                Technical Abstract: 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.  This assay was used to quantify the dose-dependent inhibition of protein synthesis in Vero-d2EGFP cells exposed to a Stx1/2-containing E. coli O157 culture supernatant or to purified Stx2.  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.


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