Specific inhibition of cytotoxicity of Shiga-like toxin 1 of enterohemorrhagic Escherichia coli by gallocatechin gallate and epigallocatechin gallate

Mechanism of inhibitory action of 8 catechins and teaflavin was investigated at low concentration against Shiga-like toxin (Stx). Viability of Vero cells largely decreased in the presence of Stx1 and Stx2 preparations. Cytotoxicity of Stx1 decreased after preincubation with gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) at 100mg/L. However, the cytotoxicity of Stx2 was not inhibited by the preincubation with catechins and teaflavin tested. The inhibitory activity of GCg and EGCg at 15mg/L (0.0327mM) was investigated against Stx preparations with various concentrations. The cytotoxicity of Stx1 at the concentration ranging from 1.6 to 50ng/mL significantly reduced (p<0.01) by the preincubation of Stx1 with GCg at 15mg/L. Similarly, the cytotoxicity of Stx1 at the concentration ranging from 3.1 to 25ng/mL was significantly reduced (p<0.01) by the preincubation with EGCg at 15mg/L. In contrast, GCg and EGCg did not inhibit cytotoxicity of Stx2 at any concentrations tested. On the other hand, EGC showed no significant effects on cytotoxicity of both Stx1 and Stx2 at the same concentrations tested. The pocket sizes formed at the center of the Stx1B and Stx2B pentamers were calculated to be 778ų and 475ų, respectively. Docking simulations were conducted with EGCg positioned in the center of the pore of StxB pentamers. The docking models showed that EGCg formed 7 hydrogen bonds with side chains of amino acids faced inside the pocket of the Stx1B pentarmer with the lowest intramolecular energy (strain energy+electrostatic energy) of-0.1kcal/mol. In contrast, in the case of Stx2B pentamer, EGCg formed 6 hydrogen bonds with the lowest intramolecular energy of 5.2kcal/mol. In silico study suggested that EGCg forms more stable structure with Stx1B pentamer than Stx2B pentamer. These results indicated that both GCg and EGCg specifically inhibited cytotoxicity of Stx1 but not of Stx2.