Inhibition of cholera toxin and other AB toxins by polyphenolic compounds

Authors: CHERUBIN, PATRICK - University Of Central Florida; GARCIA, MARIA - University Of Central Florida; CURTIS, DAVID - University Of Central Florida; BRITT, CHRISTOPHER - University Of Central Florida; CRAFT JR., JOHN - University Of Houston; BURRESS, HELEN - University Of Central Florida; BERNDT, CHRIS - University Of Central Florida; REDDY, SRIKAR - University Of Central Florida; GUYETTE, JESSICA - University Of Central Florida; ZHENG, TIANYU - University Of Central Florida; HUO, QUN - University Of Central Florida; Quinones, Beatriz; BRIGGS, JAMES - University Of Houston; TETER, KEN - University Of Central Florida

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2016
Publication Date: 11/9/2016
Citation: Cherubin, P., Garcia, M.C., Curtis, D., Britt, C.B., Craft Jr., J.W., Burress, H., Berndt, C., Reddy, S., Guyette, J., Zheng, T., Huo, Q., Quinones, B., Briggs, J.M., Teter, K. 2016. Inhibition of cholera toxin and other AB toxins by polyphenolic compounds. PLoS One. doi:10.1371/journal.pone.0166477.

Interpretive Summary: Several plant and bacterial toxins, the AB toxins, share a common structural organization that consists of a catalytic A subunit and a cell-binding B subunit. AB toxins include Shiga toxin (ST) from Escherichia coli strains such as O157:H7, cholera toxin (CT) from Vibrio cholerae, heat-labile toxin (LT) from enterotoxigenic E. coli, diphtheria toxin (DT) from Corynebacterium diphtheriae, exotoxin A (ETA) from Pseudomonas aeruginosa, and ricin from the plant Ricinus communis. These toxins are released into the extracellular milieu, but they act upon targets within the eukaryotic (mammalian) cytosol. The toxins must therefore cross a membrane barrier in order to function. Some AB toxins, such as DT, access the cytosol from acidified endosomes. Other AB toxins such as ST and CT move from the plasma membrane to the endoplasmic reticulum (ER) before passage into the cytosol through a mechanism involving the quality control system of ER-associated degradation (ERAD). For both endosome and ER translocation sites, holotoxin disassembly occurs before or concurrently with A chain entry into the cytosol. Many cellular events precede A chain entry into the cytosol. These events include (i) holotoxin binding to the cell surface; (ii) holotoxin endocytosis; (iii) vesicle-mediated trafficking of the internalized holotoxin; (iv) dissociation of the A subunit from the rest of the toxin; (v) unfolding of the A chain to a translocation-competent conformation; and (vi) translocation of the A subunit through a membrane-spanning pore into the cytosol. Despite the general similarities in their host interactions, each AB toxin utilizes a distinct subset of surface receptors, intracellular trafficking/translocation mechanisms and cytosolic targets. It is therefore difficult to inhibit multiple AB toxins with a single agent for inactivation. Using a novel cell-based assay, we identified grape seed and grape pomace (skin) extracts as potent inhibitors of ST. More recently, we have reported that grape extracts also block CT intoxication of cultured cells and intestinal loops. The anti-CT properties of grape extract included (i) stripping pre-bound toxin from the cell surface; (ii) blocking the unfolding of CTA1, the isolated A1 chain of CT; (iii) disrupting the ER-to-cytosol export of CTA1; and (iv) inhibiting the catalytic activity of CTA1. Yet the extract did not affect toxin transport from the cell surface to the ER or the dissociation of CTA1 from its holotoxin. A specific subset of host-toxin interactions were thus disrupted by the application of grape extract, as opposed to a gross alteration of toxin or cellular function. Both grape seed and grape pomace extracts are sold as nutritional supplements and are generally recognized as safe. Both extracts also have known chemical compositions: they are highly enriched (5-8% dry weight) in polyphenolic compounds which exhibit medicinal properties for heart disease and other disorders. As such, we hypothesized the polyphenolic constituents of grape extract are a source of anti-toxin activity that function through the disruption of host-toxin interactions. To test this hypothesis, we first established that grape extract confers broad-spectrum cellular resistance to several different AB toxins. Toxicity assays with individual phenolic compounds then identified one compound that inhibited ricin, three that inhibited DT, four that inhibited ETA, and twelve that inhibited CT. Additional studies focused on CT found that two compounds disrupt toxin binding at the host plasma membrane, two compounds inhibit the enzymatic activity of CTA1 and four others disrupt the cytosolic activity of CTA1 without directly affecting its enzymatic function. This work sets the foundation for the development of a broad-spectrum anti-toxin therapeutic with a known chemical composition and established modes of action.

Technical Abstract: All AB-type protein toxins have intracellular targets despite an initial extracellular location. These toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against AB toxins are therefore hard to develop because the toxins use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets. We have found grape seed extract provides resistance to five different AB toxins: cholera toxin (CT), Shiga toxin (ST), ricin, diphtheria toxin, and exotoxin A. To identify individual compounds in grape extract that can inhibit the activities of these AB toxins, we used a cell culture system to screen twenty common phenolic compounds of grape extract for anti-toxin properties. Twelve compounds inhibited CT, one inhibited ricin, three inhibited diphtheria toxin, and four inhibited exotoxin A. No individual compound generated resistance against ST, although a cocktail of all 20 compounds conferred partial resistance to ST. Additional studies were performed to determine the mechanism of inhibition against CT. Two compounds inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell. Two other compounds inhibited the enzymatic activity of CT, and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CT. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT. This work will help formulate a defined mixture of polyphenolic compounds that could potentially be used as an intervention strategy against a broad range of AB toxins.