A rapid and sensitive method to measure the functional activity of Shiga toxins in human serum.

Valentina Arfilli,Gianluigi Ardissino,Domenica Carnicelli,Gaia Scavia,Maurizio Brigotti,Erminio Torresani

doi:10.3390/toxins7114564

Abstract

Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins).

Highlights

Hemolytic uremic syndrome (HUS) is the main cause of acute renal failure in early childhood and is often the sequela of enteritis caused by Shiga toxin-producing Escherichia coli (STEC), the denomination of diarrhea-associated HUS [1,2,3]
Shiga toxins (Stx) and Stx-producing Escherichia coli (STEC) have different concurring roles in the pathogenesis of STEC-related diseases: (i) bacteria are confined to the gut, and their intimate adhesion to the epithelial lining of the bowel is principally related to watery diarrhea [8,9]; (ii) toxins cross the intestinal epithelial barrier and bind to specific glycolipid receptors, namely globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) [10], expressed on the microvasculature of the gut, causing the development of bloody diarrhea [8,9]; (iii) Stx escaping the capture by intestinal endothelial cells reach the kidney through the blood stream and bind to Gb3Cer and Gb4Cer on glomerular endothelial cells; the latter phenomenon is considered of prime importance in the onset of HUS [4,8,9]
The steps required by this method are: (i) the incubation of the cell monolayer or the suspended cells with the studied compound or specimen; (ii) the addition of [3H] leucine, followed by a 1-h incubation at 37 °C; (iii) three washings with ice-cold phosphate buffered saline (PBS) or Earle’s salt solution to remove free [3H] leucine; (iv) precipitation of cellular proteins (5 min on ice) with ice-cold 10% (v/v) trichloroacetic acid (TCA); (v) two washings with ice-cold 10% (v/v) TCA, each followed by incubation for 5 min on ice; (vi) the solubilization of the denatured cellular proteins in 0.2 M KOH; and (vii) the measure of their radioactivity in a liquid-scintillation β counter

Summary

Introduction

Hemolytic uremic syndrome (HUS) is the main cause of acute renal failure in early childhood and is often the sequela of enteritis caused by Shiga toxin-producing Escherichia coli (STEC), the denomination of diarrhea-associated HUS [1,2,3]. Stx and STEC have different concurring roles in the pathogenesis of STEC-related diseases: (i) bacteria are confined to the gut, and their intimate adhesion to the epithelial lining of the bowel is principally related to watery diarrhea [8,9]; (ii) toxins cross the intestinal epithelial barrier and bind to specific glycolipid receptors, namely globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) [10], expressed on the microvasculature of the gut, causing the development of bloody diarrhea [8,9]; (iii) Stx escaping the capture by intestinal endothelial cells reach the kidney through the blood stream and bind to Gb3Cer and Gb4Cer on glomerular endothelial cells; the latter phenomenon is considered of prime importance in the onset of HUS [4,8,9]. Free Stx has been detected in sera of STEC-infected patients during the prodromal intestinal phase before the onset of HUS [22]

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