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.

Keywords: Shiga toxin-producing Escherichia coli; cleaved Shiga toxin 2a; hemolytic uremic syndrome.

Figure 1

Effect of Stx2a on cell-free protein synthesis under reducing and non-reducing conditions. The IC₅₀ values calculated from the straight lines depicted above are reported in Table 1.

Figure 2

Effect of cleaved Stx2a (0.15 nM) on cell-free protein synthesis in the presence of 12 mM DTT, or after pretreatment (pre.) with 12 or 80 mM DTT.

Figure 3

Effect of serum samples from three different healthy donors on cell-free protein synthesis in the presence or in the absence of DTT.

Figure 4

Mixed sera from three healthy donors or RNAse A were added to the cell-free translation system. (A) Mixed sera was assayed under reducing conditions in the absence or in the presence of protease (1x cOmpleteTM) or RNAse (20 U of RiboLock or Placental RNAse Inhibitor (PRI)) blockers; (B) Mixed sera was assayed in the presence of PRI under reducing and non-reducing conditions (p < 0.0001); (C) RNAse A (10 ng) was assayed giving strong inhibitory effects, the significant protective action of PRI under reducing and non-reducing conditions is shown (p < 0.0001); (D) Mixed sera were treated with protein G Sepharose and assayed. Comparison with untreated serum and protein G Sepharose eluate was performed.

Figure 5

Effect of Stx2a on cell-free protein synthesis under reducing and non-reducing conditions in the presence of mixed sera from three healthy donors and PRI. The IC₅₀ values calculated from the straight lines depicted above are reported in Table 2.

Figure 6

Effect of Stx2a on cell-free protein synthesis under reducing and non-reducing conditions. The experiments were performed in the presence of mixed sera from three healthy donors (untreated or treated with protein G Sepharose). When serum was present, PRI was added. (A) Uncleaved form of Stx2a; (B) Cleaved form of Stx2a. The IC₅₀ values calculated from the straight lines depicted above are reported in Table 1 and Table 2. Mathematical and statistical details are reported in Supplementary Materials Table S1.