Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum.

Lucrezia Rocchetti,Elisa Varrone,Domenica Carnicelli,Reinhard Würzner,Elisa Porcellini,Maurizio Brigotti,Dorothea Orth-Höller,Beatrice Munari

doi:10.3390/toxins13020094

Lucrezia Rocchetti, Elisa Varrone + Show 6 more

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https://doi.org/10.3390/toxins13020094

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Abstract

The pathogenesis of Escherichia coli-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing E. coli (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum proteins) whose interaction is crucial for the immediate outcome and for the development of this life-threatening sequela. Stx2a, associated to circulating cells (early toxemia) or extracellular vesicles (late toxemia) in blood, is considered the main pathogenic factor in the development of eHUS. Recently, it was found that the functional properties of Stx2a (binding to circulating cells and complement components) change according to modifications of the structure of the toxin, i.e., after a single cleavage of the A subunit resulting in two fragments, A1 and A2, linked by a disulfide bridge. Herein, we describe a method to be used for the detection of the cleaved form of Stx2a in the serum of STEC-infected or eHUS patients. The method is based on the detection of the boosted inhibitory activity of the cleaved toxin, upon treatment with reducing agents, on a rabbit cell-free translation system reconstituted with human ribosomes. The method overcomes the technical problem caused by the presence of inhibitors of translation in human serum that have been stalled by the addition of RNAase blockers and by treatment with immobilized protein G. This method, allowing the detection of Stx2a at concentrations similar to those found by ELISA in the blood of STEC-infected patients, could be a useful tool to study the contribution of the cleaved form of Stx2a in the pathogenesis of eHUS.

Highlights

Renal thrombotic microangiopathy is the endpoint of the pathogenesis of Escherichia coli-induced hemolytic uremic syndrome in about 5–10% of children infected with Shiga toxin-producing E. coli strains (STEC) [1,2,3]
We have recently shown that the structure of the A chain of Stx2a is mandatory for its behavior in several steps of experimental Escherichia coli-induced hemolytic uremic syndrome (eHUS) pathogenesis [12]
We employed a luminometric fractionated system [15] derived from the classic rabbit reticulocyte radioactive translating system, which resulted in higher sensitivity to whole untreated Stx2a with respect to the parent system

Summary

Introduction

Many investigations have been undertaken to find out the relative contribution to eHUS pathogenesis of the different forms of Stx2a present in blood by considering this toxin subtype as a stable unchangeable element produced by pathogenic bacterial strains. This toxin was subjected to intense scrutiny to unravel the AB5 structure, the sequence and folding, the specific glycolipid receptor (globotriaosylceramide, Gb3Cer), the intracellular routing and the enzymatic mechanism of action on ribosomes leading to irreversible halting of protein synthesis [1,2]. Stx2a holds a trypsinsensitive region (Arg-X-X-Arg) in its A chain which is recognized by furin, the specific cellular protease responsible for the cleavage of the toxin during intracellular transport [10]

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Results

Conclusion