A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

Authors: CHERUBIN, PATRICK - University Of Central Florida; Quinones, Beatriz; ELKAHOUI, SALEM - Centre De Biotechnologie; Yokoyama, Wallace - Wally; TETER, KEN - University Of Central Florida

Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2017
Publication Date: 5/7/2017
Citation: Cherubin, P., Quinones, B., Elkahoui, S., Yokoyama, W.H., Teter, K. 2017. A cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis. Methods in Molecular Biology. 1600:25-26. doi:10.1007/978-1-4939-6958-6_3.

Interpretive Summary: AB-type protein toxins are produced by numerous bacterial pathogens and some plants. These toxins contain a catalytically active A subunit and a cell-binding B subunit. The A and B moieties can encompass different regions of a single polypeptide chain or may represent distinct proteins in various stoichiometries (e.g., AB, AB2, AB5, A2B7). Many AB toxins inhibit protein synthesis through inactivation of elongation factor 2 or the 28S rRNA. Several methods can detect the toxin-induced inhibition of protein synthesis or resulting cell death. A common procedure measures the viability of intoxicated cells by dye exclusion, MTT/MTS assay, or similar protocols. This strategy can require several days of toxin exposure and often involves additional processing steps for data collection. Furthermore, there is a temporal disconnect between the inhibition of protein synthesis and the loss of cell viability. A direct method to quantify the toxin-induced inhibition of protein synthesis measures the incorporation of radiolabeled amino acids into newly synthesized proteins. This requires the handling of radioisotopes, which is laborious, potentially hazardous, and can only accommodate a limited number of samples. Quantitative luciferase-based assays have been described that are similar to the system reported here, but these systems require several preparatory and/or processing steps to enact the detection method. A recently described assay that monitors the production and secretion of acetylcholinesterase likewise requires additional processing steps for data acquisition. As an alternative to existing technologies, we developed a simple and quantitative cell-based assay for the detection of toxins that inhibit protein synthesis. A Vero cell line with constitutive expression of a destabilized variant (t1/2 = 2 h) of the enhanced green fluorescent protein (d2EGFP) is challenged with toxin for 18-24 h. Intoxicated cells degrade d2EGFP and do not replenish the lost protein due to the toxin-induced block of protein synthesis. The fluorescent signal from Vero-d2EGFP cells is accordingly lost in proportion to the applied dose of toxin. This assay provides reproducible data with minimal hands-on effort (see Note 1). The procedure does not require radioisotopes, commercial kits, or additional processing steps. A plate reader is required for reading fluorescent samples, but the only major recurring cost is the use of black-walled, clear-bottom 96-well tissue culture microplates. As described below, the non-invasive nature of the fluorescent measurement allows the Vero-d2EGFP cells to be used for additional purposes. Furthermore, the protocol can be adapted to screen for toxin inhibitors.

Technical Abstract: AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line that stably expresses d2EGFP-N1, a variant of the enhanced green fluorescent protein (EGFP). When challenged with a toxin that inhibits protein synthesis, toxin-susceptible cells degraded d2EGFP and did not produce more of the protein. Results demonstrated that incubation with ricin reduced the Vero-d2EGFP fluorescent signal 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. Grape extracts were subjected to a modified high-performance liquid chromatography method, and the polyphenolic fractions were tested in the Vero-d2EGFP cells for anti-toxin effects. A significant loss of fluorescence was recorded for Vero-d2EGFP cells challenged with toxin in the presence of fractions 1-6. However, intoxicated cells co-incubated with fraction 7 retained a stronger fluorescent signal, representing a statistically significant difference from the intoxicated control cells (p = 0.0217, Student's t test). These collective results highlight several advantages of the Vero-d2EGFP system, including (i) relatively rapid detection of toxin activity as well as inhibitors of toxin activity; (ii) high sensitivity; (iii) minimal sample handling for data acquisition; and (iv) a non-invasive/non-terminal measurement that allows the cells to be used for other purposes such as a viability assay.