Characterization of Pathogenic Mechanisms Associated with Shiga Toxin 2 Subtypes Produced by Escherichia coli Strains

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