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

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.

SUBMITTER: Rocchetti L

PROVIDER: S-EPMC7911550 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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

Rocchetti Lucrezia L Munari Beatrice B Varrone Elisa E Porcellini Elisa E Orth-Höller Dorothea D Würzner Reinhard R Carnicelli Domenica D Brigotti Maurizio M

Toxins 20210126 2

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, ...[more]

PMID: 33530614

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Project description:Escherichia coli O157:H7 is a foodborne pathogen implicated in various multistate outbreaks. It encodes Shiga toxin on a prophage, and Shiga toxin production is linked to phage induction. An E. coli strain, designated 0.1229, that amplified Stx2a production when cocultured with E. coli O157:H7 strain PA2 was identified. Growth of PA2 in 0.1229 cell-free supernatants had a similar effect, even when supernatants were heated to 100°C for 10?min, but not after treatment with proteinase K. The secreted molecule was shown to use TolC for export and the TonB system for import. The genes sufficient for production of this molecule were localized to a 5.2-kb region of a 12.8-kb plasmid. This region was annotated, identifying hypothetical proteins, a predicted ABC transporter, and a cupin superfamily protein. These genes were identified and shown to be functional in two other E. coli strains, and bioinformatic analyses identified related gene clusters in similar and distinct bacterial species. These data collectively suggest that E. coli 0.1229 and other E. coli strains produce a microcin that induces the SOS response in target bacteria. Besides adding to the limited number of microcins known to be produced by E. coli, this study provides an additional mechanism by which stx 2a expression is increased in response to the gut microflora.IMPORTANCE How the gut microflora influences the progression of bacterial infections is only beginning to be understood. Antibiotics are counterindicated for E. coli O157:H7 infections, limiting treatment options. An increased understanding of how the gut microflora directs O157:H7 virulence gene expression may lead to additional treatment options. This work identified E. coli strains that enhance the production of Shiga toxin by O157:H7 through the secretion of a proposed microcin. Microcins are natural antimicrobial peptides that target specific species, can act as alternatives to antibiotics, and mediate microbial competition. This work demonstrates another mechanism by which non-O157 E. coli strains may increase Shiga toxin production and adds to our understanding of microcins, a group of antimicrobials less well understood than colicins.

Dataset Information

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

Publications

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

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