Figure 1.
Grape extracts inhibit cholera intoxication of culture cells.
(A–B) CHO cells were incubated for 2 h (A) or 18 h (B) with various concentrations of CT in the absence or presence of grape extract before cAMP levels were quantified. (C) CHO cells were incubated for 2 h with various concentrations of CT in the absence or presence of a chemically defined phenolic cocktail before cAMP levels were quantified. (D) CHO cells were incubated for 2 h with 100 µM forskolin in the absence or presence of grape seed extract, grape pomace extract, or phenolic cocktail before cAMP levels were quantified. For all panels, data are presented as percentages of the maximal cAMP response for the experiment (i.e., the cAMP level obtained from untreated cells exposed to 100 ng/mL of CT or to 100 µM forskolin). The averages ± standard deviations of 3–4 independent experiments with triplicate samples are shown. In panels A–C, all experimental conditions were significantly different from the No Treatment control at every matched toxin concentration (1-way ANOVA, p<0.05).
Table 1.
Composition of the chemically defined phenolic cocktail.
Figure 2.
Grape extracts inhibit the diarrheatic response to CT and LT.
A ligated intestinal loop assay in neonatal pigs was used to monitor net fluid accumulation in response to an 8 h challenge with CT (A) or LT (B). Loops were injected with PBS alone or PBS containing 10 mg grape seed extract, 1 mg grape pomace extract, and/or 10 µg toxin as indicated. Data represent the averages ± standard deviations of 3–4 replicate loops in a single pig for each toxin/experiment. Statistically significant differences between loops challenged with toxin in the absence vs. presence of either grape extract were detected for both CT (p<0.001) and LT (p<0.01) by ANOVA. No significant differences were noted between (i) loops treated with PBS alone and any extract-treated loop; (ii) loops treated with seed extract and loops treated with both seed extract and toxin; or (iii) loops treated with pomace extract and loops treated with both pomace extract and toxin.
Figure 3.
Grape extracts inhibit toxin binding to the cell surface.
CHO cells were placed on ice and exposed to FITC-CTB for 30–60 min. The cells were washed with PBS to remove unbound toxin, and fluorescent output was determined with a plate reader. (A) Cells were co-incubated with FITC-CTB and grape seed or pomace extract for 60 min. (B) Cells were exposed to grape extract for 30 min. The extracts were removed by washing with PBS, and the cells were then exposed to FITC-CTB for 30 min. (C) Cells were exposed to FITC-CTB for 30 min. Unbound toxin was removed by washing with PBS, and the cells were then exposed to grape extract for 30 min. (D) A mixture of FITC-CTB and grape extract was placed in a 3500 MWCO dialysis cup. After an overnight dialysis, FITC-CTB was applied to cells for 60 min. All steps of all experiments were performed at 4°C, and all experiments contained a No Treatment control in which FITC-CTB was not exposed to grape extract but was otherwise treated identically to the extract-treated samples. For all panels, data are presented as percentages of the maximal FITC-CTB signal obtained from the No Treatment control. The means ± standard errors of the means of 4 independent experiments with 6 replicates per condition are shown. Asterisks denote statistically significant differences between untreated and extract-treated cells (1-way ANOVA, p<0.01).
Figure 4.
Grape extracts confer protection against CT after endocytosis of the toxin.
CHO cells were incubated with CT for 30 min at 4°C. Unbound toxin was removed, and the cells were warmed to 37°C. Grape seed extract, grape pomace extract, or BfA was added to the cells at the time of warming to 37°C (0 min post-toxin incubation) and 15, 30, or 60 min after warming to 37°C. cAMP levels were determined 2 h after the initial warming to 37°C. The maximal cAMP response was obtained from toxin-treated cells incubated in the absence of extract or BfA. The averages ± standard deviations of 3 independent experiments with triplicate samples are shown. At all time intervals, the differences between untreated cells and extract- or BfA-treated cells were statistically significant (1-way ANOVA, p<0.01).
Figure 5.
Grape seed extract does not prevent toxin transport to the ER.
HeLa cells were incubated with CT for 30 min at 4°C. Unbound toxin was removed, and the cells were warmed to 37°C. Grape seed extract was only added to the cells 15, 30, or 60 min after warming to 37°C. As additional controls, cells were untreated after warming (Un) or were exposed to BfA at the time of warming (BfA). Cell extracts generated after the 4°C pulse labeling (P) or after a total of 2 h at 37°C were separated into membrane and cytosolic fractions. The membrane fractions were resolved by non-reducing SDS-PAGE before Western blot analysis with an anti-CTA1 antibody. Reduction of the CTA1/CTA2 disulfide bond is indicative of toxin transport to the ER.
Figure 6.
Grape seed extract does not prevent PDI-mediated disassembly of the CT holotoxin.
Reduced PDI and grape seed extract were perfused over an SPR sensor coated with the CT holotoxin. The PDI- and extract-containing perfusion buffer was replaced with a buffer containing an anti-CTA1 antibody (first arrowhead), which in turn was replaced with a buffer containing an anti-KDEL antibody (second arrowhead). At the beginning of the experiment, the signal corresponding to the mass of the intact CT holotoxin was set at a baseline RIU value of zero (dotted line).
Figure 7.
Grape extracts strip CT from lipid bilayers.
At time point 0, CT was perfused over a SPR sensor coated with GM1-containing LUVs. Grape seed or grape pomace extract was added to the perfusion buffer at 200 sec in the continued presence of CT. One of two representative experiments is shown.
Figure 8.
Grape extracts prevent the temperature-induced unfolding of CTA1.
(A) A purified CTA1/CTA2 heterodimer was placed in 20 mM sodium phosphate buffer (pH 7.4) containing 10 mM β-mercaptoethanol. Aliquots (1 µg) of the toxin were either left untreated, treated with grape seed extract, or treated with grape pomace extract as indicated. All samples were incubated at the indicated temperatures for 1 h. The toxins were then shifted to 4°C and exposed to the thermolysin protease for 1 h. Samples were resolved by SDS-PAGE and Coomassie staining, which does not visualize the 5 kDa CTA2 subunit. (B) Purified α-casein was placed in 20 mM sodium phosphate buffer (pH 7.4) containing 10 mM β-mercaptoethanol. Aliquots (5 µg) of the protein were incubated from 1 h at 4°C in the absence or presence of thermolysin before visualization by SDS-PAGE and Coomassie staining. Samples exposed to thermolysin were untreated or co-incubated with either grape seed or grape pomace extract as indicated.
Figure 9.
Grape extracts prevent CTA1 translocation to the cytosol.
HeLa cells were incubated with CT for 30 min at 4°C. Unbound toxin was removed, and the cells were warmed to 37°C. Grape pomace (A) or grape seed (B) extract was added to the cells 15, 30, or 60 min after warming to 37°C. Cell extracts generated after a total of 2 h at 37°C were separated into membrane and cytosolic fractions, and the cytosolic fractions were perfused over an SPR sensor coated with an anti-CTA1 antibody. The cytosolic fraction from unintoxicated cells was used as a negative control, and CTA standards were used as positive controls. All samples were perfused over the same SPR sensor; the data is presented in two panels for clarity. One of two representative experiments is shown.
Figure 10.
Grape extracts do not prevent the secretion of free CTA1.
HeLa cells were incubated with CT for 30 min at 4°C. Unbound toxin was removed, and the cells were warmed to 37°C. BfA was added immediately after warming, and grape pomace or grape seed extract was added to the cells 15 min after warming to 37°C. Media samples collected after a total of 2 h at 37°C were perfused over an SPR sensor coated with an anti-CTA1 antibody. The extracellular medium from unintoxicated cells was used as a negative control, and CTA standards were used as positive controls. One of two representative experiments is shown.
Figure 11.
Grape extracts inhibit the ADP-ribosyltransferase activity of CTA1.
(A) Dilutions of CTA1 mixed with DEA-BAG in the presence or absence of grape extract were placed at 25°C for 2 h. The ADP-ribosylation of DEA-BAG was then assessed by fluorometry; increasing fluorescent units correspond to increasing levels of substrate modification. Data are presented as the averages ± ranges of two replicate samples per condition. One of two representative experiments is shown. (B) DEA-BAG was placed in buffer lacking or containing either grape seed or grape pomace extract. The intrinsic fluorescence of DEA-BAG and extract-treated DEA-BAG was then assessed by fluorometry. Data are presented as percentages of the value obtained from untreated DEA-BAG, which was used at a concentration that did not saturate signal detection. The averages ± standard deviations of 3 independent experiments with triplicate samples are shown.