T cell Receptor Vβ9 in Method for Rapidly Quantifying Active Staphylococcal Enterotoxin Type-A without Live Animals

Paula Do,Reuven Rasooly,Bradley Hernlem,Xiaohua He

doi:10.3390/toxins11070399

Abstract

Staphylococcal food poisoning is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus. Staphylococcal enterotoxin type A (SEA) is the predominant toxin produced by S. aureus strains isolated from food-poisoning outbreak cases. For public safety, assays to detect and quantify SEA ideally respond only to the active form of the toxin and this usually means employing disfavored live animal testing which suffers also from poor reproducibility and sensitivity. We developed a cell-based assay for SEA quantification in which biologically-active SEA is presented by Raji B-cells to CCRF-CEM T-cells resulting in internalization of Vβ9 within 2 hours with dose dependency over a 6-log range of SEA concentrations. This bioassay can discern biologically active SEA from heat-inactivated SEA and is specific to SEA with no cross reactivity to the homologically-similar SED or SEE. In this study, we terminated any ongoing biochemical reactions in accessory cells while retaining the morphology of the antigenic sites by using paraformaldehyde fixation and challenged the current model for mechanism of action of the SEA superantigen. We demonstrated for the first time that although fixed, dead accessory cells, having no metabolic functions to process the SEA superantigen into short peptide fragments for display on their cell surface, can instead present intact SEA to induce T-cell activation which leads to cytokine production. However, the level of cytokine secretion induced by intact SEA was statistically significantly lower than with viable accessory cells, which have the ability to internalize and process the SEA superantigen.

Highlights

Staphylococcus aureus is a prevalent bacterial pathogen that produces a wide variety of exoproteins that cause various types of disease
Superantigenic activity and the gastroenteric syndrome are two separate functions of staphylococcal enterotoxins (SEs), there is a high correlation between these activities
The data presented in this study show, for the first time, that within 2 hours after stimulation with Staphylococcal enterotoxin type A (SEA), there is internalization of T-cell receptor (TCR) Vβ9 as demonstrated by the reduction of TCR Vβ9 surface expression within a single T-cell line, and this phenomenon can be used for rapid detection and quantification of biologically-active SEA

Summary

Introduction

Staphylococcus aureus is a prevalent bacterial pathogen that produces a wide variety of exoproteins that cause various types of disease. Pathogenesis is mediated by virulence factors including some 23 different staphylococcal enterotoxins (SEs) that induce gastroenteric syndrome, exhibit emetic activity, and are the causative agents of food poisoning affecting 241,148 persons annually in the United. Some of these SE subtypes are active at very low concentration, as small as 1 fg/mL [2,3]. SEs function as superantigens that activate CD4+ T cells, cause proliferation of T-cells in a dose and time dependent manner [4], and induce differential regulation of CD154 [5] that is responsible for costimulatory signals to B cells. Superantigenic activity and the gastroenteric syndrome are two separate functions of SEs, there is a high correlation between these activities.

Methods

Results

Conclusion