Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum
The pathogenesis of <i>Escherichia coli</i>-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing <i>E. coli</i> (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum prote...
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doaj-b72a2db17c634717aa321f3e603c80112021-01-27T00:06:27ZengMDPI AGToxins2072-66512021-01-0113949410.3390/toxins13020094Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human SerumLucrezia Rocchetti0Beatrice Munari1Elisa Varrone2Elisa Porcellini3Dorothea Orth-Höller4Reinhard Würzner5Domenica Carnicelli6Maurizio Brigotti7Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, ItalyDepartment of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, ItalyDepartment of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, ItalyDepartment of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, ItalyInstitute of Hygiene and Medical Microbiology, Medical University of Innsbruck, A-6020 Innsbruck, AustriaInstitute of Hygiene and Medical Microbiology, Medical University of Innsbruck, A-6020 Innsbruck, AustriaDepartment of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, ItalyDepartment of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, ItalyThe pathogenesis of <i>Escherichia coli</i>-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing <i>E. coli</i> (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.https://www.mdpi.com/2072-6651/13/2/94hemolytic uremic syndromecleaved Shiga toxin 2aShiga toxin-producing <i>Escherichia coli</i> |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Lucrezia Rocchetti Beatrice Munari Elisa Varrone Elisa Porcellini Dorothea Orth-Höller Reinhard Würzner Domenica Carnicelli Maurizio Brigotti |
spellingShingle |
Lucrezia Rocchetti Beatrice Munari Elisa Varrone Elisa Porcellini Dorothea Orth-Höller Reinhard Würzner Domenica Carnicelli Maurizio Brigotti Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum Toxins hemolytic uremic syndrome cleaved Shiga toxin 2a Shiga toxin-producing <i>Escherichia coli</i> |
author_facet |
Lucrezia Rocchetti Beatrice Munari Elisa Varrone Elisa Porcellini Dorothea Orth-Höller Reinhard Würzner Domenica Carnicelli Maurizio Brigotti |
author_sort |
Lucrezia Rocchetti |
title |
Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum |
title_short |
Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum |
title_full |
Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum |
title_fullStr |
Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum |
title_full_unstemmed |
Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum |
title_sort |
method for the detection of the cleaved form of shiga toxin 2a added to normal human serum |
publisher |
MDPI AG |
series |
Toxins |
issn |
2072-6651 |
publishDate |
2021-01-01 |
description |
The pathogenesis of <i>Escherichia coli</i>-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing <i>E. coli</i> (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. |
topic |
hemolytic uremic syndrome cleaved Shiga toxin 2a Shiga toxin-producing <i>Escherichia coli</i> |
url |
https://www.mdpi.com/2072-6651/13/2/94 |
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