Fighting Shigella by Blocking Its Disease-Causing Toxin

Abstract

Shiga toxin is an AB 5 toxin produced by Shigella species, while related toxins are produced by Shiga toxin-producing Escherichia coli (STEC). Infection by Shigella can lead to bloody diarrhea followed by the often fatal hemolytic uremic syndrome (HUS). In the present paper, we aimed for a simple and effective toxin inhibitor by comparing three classes of carbohydrate-based inhibitors: glycodendrimers, glycopolymers, and oligosaccharides. We observed a clear enhancement in potency for multivalent inhibitors, with the divalent and tetravalent compounds inhibiting in the millimolar and micromolar range, respectively. However, the polymeric inhibitor based on galabiose was the most potent in the series exhibiting nanomolar inhibition. Alginate and chitosan oligosaccharides also inhibit Shiga toxin and may be used as a prophylactic drug during shigella outbreaks.

Nanobody-Based Bispecific Neutralizer for Shiga Toxin-Producing E. coli

“…Next, we tested the ability of this bispecific construct for functionally neutralizing Shiga toxin and Intimin. Inhibition of Stx2B binding to its receptor Gb3 trisaccharide was determined by using a functional ELISA type assay as we described previously . The Gb3 construct was immobilized on the plate and the Bio-Stx2B binding to Gb3 was determined in the presence or absence of siNb (Figure A left panel, and Figure S6A).…”

“…Inhibition of Stx2B binding to its receptor Gb3 trisaccharide was determined by using a functional ELISA type assay as we described previously. 15 The Gb3 construct was immobilized on the plate and the Bio-Stx2B binding to Gb3 was determined in the presence or absence of siNb (Figure 3A left panel, and Figure S6A). As shown in Figure 3C, the addition of BSA as a control had no effect on the Bio-Stx2B binding, while introduction of siNb blocked that in a concentration dependent manner, with an EC 50 of 133 nM.…”

“…The inhibitory effect of nanobodies on the binding of Stx2B to Gb3 was determined using an ELISA type functional assay essentially as we described previously . Briefly, a 96-well plate (Nunc PolySorb) were coated with FSL-Gb3 (5 μg/mL, 100 μL/well) in PBS overnight at 4 °C.…”

“…The inhibitory effect of nanobodies on the binding of Stx2B to Gb3 was determined using an ELISA type functional assay essentially as we described previously. 15 Briefly, a 96-well plate (Nunc PolySorb) were coated with FSL-Gb3 (5 μg/mL, 100 μL/ well) in PBS overnight at 4 °C. The unbound FSL-Gb3 was removed by washing the plate three times with PBS and 200 μL of 3% BSA in PBS was added into each well to block the potential unspecific binding sites at room temperature for 2 h. After brief washing with PBS, the Gb3-coated plate was incubated with 100 μL of Bio-Stx2B (1000 nM) alone as positive control or Bio-Stx2B preincubated with the bispecific nanobody for 0.5 h at concentration as indicated in the context.…”

“…After entering the host cells, the A subunit matures as an active N -glycosidase that cleaves an adenine from the 28S RNA in the ribosome and interferes with proteins synthesis which results in cell death . With respect to neutralizing Shiga toxin-caused cytotoxicity, most attempts have been focused on developing Gb3 competitive inhibitors, for example multivalent neutralizers by displaying the functional carbohydrate part of Gb3 or its analogues on a probiotic, nanoparticle, polymer, or multivalent scaffold. − Passive immunization including conventional antibodies and recombinant antibody fragments such as ScFv and Fab have also been developed to target and neutralize Shiga toxins. − Interestingly, nanobodies, the variable domain of camelid heavy chain antibody, against Stx1 and Stx2 as neutralizer also showed therapeutic potential for HUS. − Inherent advantages of a nanobody over conventional antibody such as easy production, high stability, and tolerance to modifications have made it a promising alternative for targeted-drug development targeting infectious diseases. − …”