Inhibition of Toxin Translocation Can Reverse Cholera and ETEC-Mediated Diarrhea

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DESCRIPTION (provided by applicant): Cholera toxin (CT) and heat-labile toxin (LT) from enterotoxigenic Escherichia coli (ETEC) are highly related AB5-type protein toxins composed of an enzymatic A1 subunit, an A2 linker, and a cell-binding B pentamer. Both toxins generate a diarrheatic response through the ADP-ribosylation of a host protein, Gs¿. CT and LT attack Gs¿ within the cytosol of the host cell, but they are initially secreted into the extracellular medium. The toxins must therefore cross a membrane barrier in order to function. This only occurs after the intact holotoxin moves by vesicle carriers from the cell surface through multiple compartments of the host endomembrane system en route to the endoplasmic reticulum (ER). The A1 subunit dissociates from the rest of the toxin in the ER, unfolds, and passes through a protein-conducting channel to enter the cytosol. The translocated A1 subunit then interacts with host factors to regain a functional conformation for the activation of Gs¿. It is believed that recovery from cholera and ETEC-mediated diarrhea can only occur after intoxicated enterocytes, which have a 3-5 day lifespan, are sloughed from the intestinal epithelium. A number of observations indicate this is not the case. We have recently shown that a threshold concentration of cytosolic CTA1 is required to elicit a cytopathic effect. We have also identified two therapeutic agents that block toxin translocation to the cytosol, and we have documented the ubiquitin-independent proteasomal degradation of cytosolic CTA1. Other reports indicate it is possible to de-activate the ADP-ribosylated form of Gs?. These collective observations strongly suggest that cholera and ETEC-mediated diarrhea are reversible events. If CTA1/LTA1 translocation to the cytosol is blocked after toxin exposure, the pool of toxin already in the cytosol will be degraded and will not be replenished. The cytosolic pool of CTA1/LTA1 will therefore drop below the requisite threshold concentration, allowing the host cell to de-activate Gs? and recover from intoxication. To test this model, we will use a novel toxin detection system based on surface plasmon resonance (SPR) technology to quantify holotoxin and A1 subunit distributions in the cytosol, endomembrane system, and extracellular medium over the lifespan of an intoxicated enterocyte. These parameters have not been determined for any AB toxin. Based on published and preliminary data, we predict the endomembrane system serves as a long-term reservoir for the slow but continual delivery of CTA1/LTA1 to the cytosol. We further predict the low levels of CTA1/LTA1 which reach the cytosol, combined with cellular mechanisms to clear the cytosol of toxin and reverse the effects of intoxication, will permit treatment and recovery from CT/LT intoxication. This will be tested with toxicity assays and SPR-based assays to monitor the delivery and persistence of cytosolic toxin in cells treated with drugs that block toxin translocation. Validation of our model will provide a molecular basis for new post-exposure therapeutic strategies focused on the translocation event. HEALTH RELEVANCE: Vibrio cholerae and enterotoxigenic Escherichia coli produce highly related toxins that enter our intestinal cells to induce a potentially fatal case of diarrhea. The amounts of intracellular toxin needed to initiate and sustain a diarrheatic response are unknown. Here, we will establish these parameters and demonstrate recovery from intoxication is possible with treatments that lower the levels of intracellular toxin.