Abstract
Cholera pathogenesis occurs due to synergistic pro-secretory effects of several toxins, such as cholera toxin (CTX) and Accessory cholera enterotoxin (Ace) secreted by Vibrio cholerae strains. Ace activates chloride channels stimulating chloride/bicarbonate transport that augments fluid secretion resulting in diarrhea. These channels have been targeted for drug development. However, lesser attention has been paid to the interaction of chloride channel modulators with bacterial toxins. Here we report the modulation of the structure/function of recombinant Ace by small molecule calcium-activated chloride channel (CaCC) inhibitors, namely CaCCinh-A01, digallic acid (DGA) and tannic acid. Biophysical studies indicate that the unfolding (induced by urea) free energy increases upon binding CaCCinh-A01 and DGA, compared to native Ace, whereas binding of tannic acid destabilizes the protein. Far-UV CD experiments revealed that the α-helical content of Ace-CaCCinh-A01 and Ace-DGA complexes increased relative to Ace. In contrast, binding to tannic acid had the opposite effect, indicating the loss of protein secondary structure. The modulation of Ace structure induced by CaCC inhibitors was also analyzed using docking and molecular dynamics (MD) simulation. Functional studies, performed using mouse ileal loops and Ussing chamber experiments, corroborate biophysical data, all pointing to the fact that tannic acid destabilizes Ace, inhibiting its function, whereas DGA stabilizes the toxin with enhanced fluid accumulation in mouse ileal loop. The efficacy of tannic acid in mouse model suggests that the targeted modulation of Ace structure may be of therapeutic benefit for gastrointestinal disorders.
Highlights
The diarrheal disease cholera caused by Gram negative bacteria Vibrio cholerae is still a potential threat in many developing countries [1]
Similar effect was found with high concentration of tannic acid, while low concentration of the same was not able to inhibit basal as well as Accessory cholera enterotoxin (Ace) stimulated increases short circuit current (Isc) (Fig 5B). These results demonstrate that low concentration of CaCCinh-A01 and tannic acid has nothing to do with calcium-activated chloride channel (CaCC) inhibition, rather they altered the structure of Ace, which in turn affected the degree of fluid accumulation in mouse ileal loop experiments (Fig 6)
Activation of chloride channel(s) and inhibition of electroneutral sodium-chloride absorption is a pivotal step in the pathogenesis of secretory diarrhea, caused by V. cholerae infection [32]
Summary
The diarrheal disease cholera caused by Gram negative bacteria Vibrio cholerae is still a potential threat in many developing countries [1]. Pathogenesis of cholera occurs due to synergistic effect of a number of toxins produced by V. cholerae [2, 3]. Ace is known to activate calcium-dependent chloride-bicarbonate secretion in human colonic carcinoma T84 cell line [6]. We have previously reported that Ace potentiated ATP stimulated chloride secretion in T84 cells via stimulation of calcium-activated chloride channel (CaCC) [7]. Ubiquitous expression of CaCCs in both epithelial and non-epithelial cells, along with their involvement in broad range of biological functions, especially fluid secretion in intestinal cells, make CaCCs potential drug targets for secretory diarrhea [8]
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