E. coli heat labile toxin (LT) inactivation by specific polyphenols is aggregation dependent

Recently, polyphenol extracts were suggested to inhibit binding of Escherichia coli heat labile enterotoxin (LT) to its intestinal receptor GM1. Therefore, polyphenols are promising feed or food supplements to combat enterotoxigenic infections. Little is known of the precise mechanism, or the type of polyphenol required. Here, seven different polyphenols were tested in vitro (1) for inhibition of LT binding to GM1 (GM1-ELISA), (2) for LT inhibitory activity in the cAMP Vero-cell assay, and (3) by testing the aggregating properties of polyphenols with LT using molecular weight exclusion membrane filters, and by centrifugation techniques. Results showed only three out of seven polyphenols, pentagalloylglucose (PGG), epigallocatechingallate (EGCG) and gallocatechingallate (GCG), to effectively inhibit binding of LT to GM1, and to inhibit induction of cAMP in Vero cells, and that PGG is the most effective. Blocking of the GM1 receptor is unlikely as a mechanism because pre-incubation of GM1 with polyphenols had no effect. Co-incubation of polyphenols with forskolin did not interfere with cAMP production in Vero cells, showing that polyphenol activity is not directly related to cAMP. It is concluded that the inhibitory activities of these three polyphenols may coincide with the formation of large (>100 kDa) LT-polyphenol aggregates. Enterotoxin inactivation appears to require a minimum of two galloyl moieties in polyphenol structure and the pentagalloyl PGG is the most effective.