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

Arfilli V, Carnicelli D, Ardissino G, Torresani E, Scavia G, Brigotti M - Toxins (Basel) (2015)

Bottom Line: 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.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.Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Sede di Patologia Generale, Università di Bologna, Via San Giacomo 14, 40126 Bologna, Italy. arfilli.v@libero.it.

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).

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Detection of Stx through the radioactive protein synthesis assay with Raji cells. The old procedure was point-by-point described in the text and modified as follows: (A) no centrifugation runs, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (B) single centrifugation run, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (C) single centrifugation run, addition of a filtration step of the denatured proteins upon treatment of cells with KOH. Blank values were obtained by addition of the labeled amino acid on ice at the end of the incubation steps. Where indicated, 150 pM Stx1a was added. *p < 0.05 as compared to control cells (t-test).
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toxins-07-04564-f001: Detection of Stx through the radioactive protein synthesis assay with Raji cells. The old procedure was point-by-point described in the text and modified as follows: (A) no centrifugation runs, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (B) single centrifugation run, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (C) single centrifugation run, addition of a filtration step of the denatured proteins upon treatment of cells with KOH. Blank values were obtained by addition of the labeled amino acid on ice at the end of the incubation steps. Where indicated, 150 pM Stx1a was added. *p < 0.05 as compared to control cells (t-test).

Mentions: The first objective of our study was the realization of a technically-sound procedure composed of the lowest number of steps required to achieve the abovementioned goals. We chose Raji cells, which express Gb3Cer [40], the specific receptor for Stx, and show a very quick response to these bacterial toxins, since protein synthesis was completely inhibited after a 3-h incubation with picomolar concentrations of Stx [31]. To simplify the procedure described above, the determination of protein synthesis inhibition in Raji cells challenged with Stx1a (150 pM for 3 h) was performed with the following modifications: (i) the centrifugation steps were omitted; (ii) a further incubation (10 min at 90 °C) of the TCA-precipitated proteins was introduced; and (iii) TCA-precipitated proteins were collected on glass microfiber filters (GF/C Whatman). The incubation step was required in order to cleave the bond between tRNAs and amino acids, thus reducing background values (blank). As shown in Figure 1A, protein synthesis was detectable and significantly inhibited by Stx, even though the protocol showed a main drawback, since the filtration step required several hours due to the huge amount of proteins present in the fetal bovine serum (FBS) added to the cell culture medium. To circumvent this problem, a single centrifugation run was introduced at the end of the incubation with toxins to eliminate the excess of proteins. Raji cells were resuspended in PBS and the procedure repeated as described above. The results were quite similar (Figure 1B), but in this case, the filtration time was very short (less than 2 min per sample). Finally, the definitive protocol was further simplified since cells were resuspended in 0.1 M KOH instead of in PBS. This allowed the removal of the incubation step at 90 °C, because the bonds connecting amino acids and tRNAs are broken by the alkaline pH at room temperature. Protein synthesis was efficiently measured and completely inhibited by Stx1a (Figure 1C). In conclusion, almost identical results have been obtained with the different approaches shown in Figure 1, even though the simpler and most rapid method is that in panel C (detailed description in Scheme 1).


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

Arfilli V, Carnicelli D, Ardissino G, Torresani E, Scavia G, Brigotti M - Toxins (Basel) (2015)

Detection of Stx through the radioactive protein synthesis assay with Raji cells. The old procedure was point-by-point described in the text and modified as follows: (A) no centrifugation runs, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (B) single centrifugation run, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (C) single centrifugation run, addition of a filtration step of the denatured proteins upon treatment of cells with KOH. Blank values were obtained by addition of the labeled amino acid on ice at the end of the incubation steps. Where indicated, 150 pM Stx1a was added. *p < 0.05 as compared to control cells (t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663520&req=5

toxins-07-04564-f001: Detection of Stx through the radioactive protein synthesis assay with Raji cells. The old procedure was point-by-point described in the text and modified as follows: (A) no centrifugation runs, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (B) single centrifugation run, addition of a filtration step upon incubation at 90 °C of the denatured proteins; (C) single centrifugation run, addition of a filtration step of the denatured proteins upon treatment of cells with KOH. Blank values were obtained by addition of the labeled amino acid on ice at the end of the incubation steps. Where indicated, 150 pM Stx1a was added. *p < 0.05 as compared to control cells (t-test).
Mentions: The first objective of our study was the realization of a technically-sound procedure composed of the lowest number of steps required to achieve the abovementioned goals. We chose Raji cells, which express Gb3Cer [40], the specific receptor for Stx, and show a very quick response to these bacterial toxins, since protein synthesis was completely inhibited after a 3-h incubation with picomolar concentrations of Stx [31]. To simplify the procedure described above, the determination of protein synthesis inhibition in Raji cells challenged with Stx1a (150 pM for 3 h) was performed with the following modifications: (i) the centrifugation steps were omitted; (ii) a further incubation (10 min at 90 °C) of the TCA-precipitated proteins was introduced; and (iii) TCA-precipitated proteins were collected on glass microfiber filters (GF/C Whatman). The incubation step was required in order to cleave the bond between tRNAs and amino acids, thus reducing background values (blank). As shown in Figure 1A, protein synthesis was detectable and significantly inhibited by Stx, even though the protocol showed a main drawback, since the filtration step required several hours due to the huge amount of proteins present in the fetal bovine serum (FBS) added to the cell culture medium. To circumvent this problem, a single centrifugation run was introduced at the end of the incubation with toxins to eliminate the excess of proteins. Raji cells were resuspended in PBS and the procedure repeated as described above. The results were quite similar (Figure 1B), but in this case, the filtration time was very short (less than 2 min per sample). Finally, the definitive protocol was further simplified since cells were resuspended in 0.1 M KOH instead of in PBS. This allowed the removal of the incubation step at 90 °C, because the bonds connecting amino acids and tRNAs are broken by the alkaline pH at room temperature. Protein synthesis was efficiently measured and completely inhibited by Stx1a (Figure 1C). In conclusion, almost identical results have been obtained with the different approaches shown in Figure 1, even though the simpler and most rapid method is that in panel C (detailed description in Scheme 1).

Bottom Line: 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.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.Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, Sede di Patologia Generale, Università di Bologna, Via San Giacomo 14, 40126 Bologna, Italy. arfilli.v@libero.it.

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).

Show MeSH
Related in: MedlinePlus