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Shiga toxins produced by enterohemorrhagic Escherichia coli and other pathogenic strains of E. coli are major virulence factors. Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) are released into the extracellular space but inactive ribosomes inside the host cell, which eventually leads to mammalian cell death. Epidemiological studies have shown that Stx2 is more frequently linked to severe illness than Stx1. Moreover, there are multiple subtypes of Stx2, and they all display differential in vivo toxicities in the mammalian host cell. We hypothesize in vivo toxin potency is linked to the extent of toxin accumulation in the cytosol of the host cell. Furthermore, we hypothesize Stx2 must accumulate and persist in the host cytosol to maintain the effects of intoxication. The goals of this project, outlined in this agreement, will test these predictions and provide a foundation for new intervention strategies that could be used to inactivate Stx2. The first goal of this Agreement will establish the efficiency of toxin delivery from the cell surface to the cytosol for the different Stx2 subtypes. We hypothesize a direct correlation between the in vivo potency of a Stx2 subtype and the efficiency of its delivery to the cytosol of the host cell. Using a novel assay developed in Professor Teter's lab at the University of Central Florida, we will quantify the accumulation of cytosolic toxin over time. This will allow us to calculate, for the first time, how many Stx2 molecules are in the cytosol of an intoxicated mammalian cell. When compared to the total quantity of Stx2 associated with the cell surface of the host cell, the efficiency of toxin delivery to the cytosol for a particular Stx2 subtype can then be quantified. By correlating the amount of cytosolic toxin to the extent of ribosome inactivation, the expected results obtained in this project should be able to determine how many molecules of cytosolic toxin are required to elicit a cellular effect. The second goal of this Agreement will determine whether polyphenolic compounds from grape extracts can prevent toxin delivery to the host cytosol and thereby reverse the cellular effects of intoxication. We hypothesize that Stx2 must accumulate and persist in the host cytosol to maintain the effects of intoxication, so the extract-induced inhibition is a result of a reduced toxin delivery to the cytosol. Consequently, this effect allows cellular recovery from exposure to various Stx2 subtypes. An expansion of this project will focus on the identification of the individual active anti-toxin component(s) of grape extract. The expected results of this Agreement will provide proof-of-principle for novel intervention strategies involving toxin inactivation through the use of plant polyphenolic compounds. The foundation for this strategy will be provided by studies that (i) provide a molecular basis for differential in vivo potency of the Stx2 subtypes; (ii) quantify how much toxin must reach the host cell cytosol to initiate a cellular effect; and (iii) demonstrate grape extract can preventing further toxin access to the cytosol
The efficiency of the delivery of Shiga toxin 2 (Stx2), produced by pathogenic Escherichia coli, from the cell surface to the cytosol has never been calculated. The number of cytosolic toxin molecules required to elicit the effect in the mammalian host cell is also unknown. These long-standing questions are directly relevant to the development of intervention strategies that could be effective after toxin exposure. The proposed hypothesis states the extent of intoxication in the host cell cytosol is directly related to the toxin amount as well as the particular subtypes of Stx2. In addition, the prediction is that Stx2 must reach a threshold concentration in the host cell cytosol to initiate a cellular effect. Because the cytosolic pool of toxin is degraded, it must be continually replenished to maintain the effects of intoxication. An inhibition of further toxin delivery to the cytosol after the onset of intoxication could therefore promote recovery from intoxication: the existing pool of cytosolic toxin would be degraded and would not be replenished, thus lowering the amount of cytosolic toxin to the point where it is no longer effective. The proposed work for this Agreement will involve a collaboration with Professor Teter?s lab, which has developed a novel, quantitative method to monitor toxin accumulation in the host cytosol. With this method, toxin-challenged mammalian cells are separated into distinct cytosolic and membrane/organelle fractions at defined post-exposure intervals. Toxin present in either fraction is detected and quantified using the highly-sensitive method of surface plasmon resonance. The efficiency of toxin delivery to the cytosol can be determined by comparing the amount of cytosolic toxin to the total cell-associated quantity of toxin. Our assay will be used to complete both project goals: (i) establish a direct correlation between the in vivo potency of a Stx2 variant and the efficiency of its delivery to the host cytosol; and (ii) demonstrate whether polyphenolics in grape extract will facilitate toxin inactivation by reducing the cytosolic quantity of Stx2 below its effective concentration. The goals of this Agreement will require a multi-year effort. Quantitative analysis requires each assay to be run several times, and data processing for each experiment involves substantial effort since multiple subtypes of Stx2 will be examined. The assays will also utilize intestinal Caco-2 cells that can be grown as a polarized epithelial monolayer. This will mimic physiological conditions; however, Caco-2 cells grow slowly and require some time to form a polarized monolayer. This project will address key questions regarding Stx2 pathogenesis and the cell biology of intoxication with Stx2. The results of our investigations will provide a molecular basis for the differential toxicity of various Stx2 subtypes and explore the use of grape extract as an alternative intervention method for the inactivation of Stx2. These goals are consistent with USDA?s mission in elucidating the molecular mechanisms of bacterial pathogenesis and developing new methods for the inactivation of food-borne pathogens.