Inhibition of Bacterial and Plant AB Toxins by Polyphenolic Compounds

Introduction: The AB-type protein toxins elicit a cytotoxic effect by inhibiting protein synthesis. AB-type toxins include Shiga toxins from Escherichia coli and ricin from the plant Ricinus communis.  Several methods have been developed to detect the toxin-induced inhibition of protein synthesis but are limited by complex and laborious processing steps.

Purpose:  Given the limitations of current methods, the present study develop a simple, rapid and quantitative cell-based assay to detect the inhibition of protein synthesis by AB-type toxins.

Methods:  A Vero cell line, Vero-d2EGFP, was constructed to constitutively express a destabilized variant of the enhanced green fluorescent protein (EGFP).  This destabilized EGFP variant was employed as a sensitive marker for measuring the inhibition of protein synthesis.  To screen for natural compounds that can inactivate AB toxins, grape extracts were subjected to separation and purification to different degrees of polymerization by using high-performance liquid chromatography.

Results: Incubation with ricin reduced the Vero-d2EGFP fluorescence in a dose-dependent manner.  The loss of EGFP fluorescence was much more dramatic than the loss of cell viability:  a half-maximal effective ricin concentration (ED50) of 0.03 ng/mL was recorded by the Vero-d2EGFP assay, whereas cell viability assay reported an ED50 of 0.7 ng/mL.  The Vero-d2EGFP assay was then employed to screen for toxin inhibitors.  A significant loss of fluorescence was recorded for Vero-d2EGFP cells challenged with Shiga toxins in the presence of fractions 1-6, composed of mostly polyphenolic monomers and dimers.  However, intoxicated cells co-incubated with fraction 7, composed of mostly polyphenolic tetramers, retained a stronger fluorescent signal, representing a statistically significant difference from the intoxicated control Vero-d2EGFP cells (p = 0.0217, Student's t test).

Significance: These results highlight that the Vero-d2EGFP assay is highly sensitive and enables the rapid detection and inactivation of AB5 toxin activity with minimal sample handling for data acquisition.