Oral Therapeutic Agents with Highly Clustered Globotriose for Treatment of Shiga ToxigenicEscherichia coliInfections

Abstract

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

Transmembrane BAX Inhibitor Motif Containing (TMBIM) Family Proteins Perturbs a trans-Golgi Network Enzyme, Gb3 Synthase, and Reduces Gb3 Biosynthesis

“…Preparation of Shiga Toxin 1 (Stx1) and Fluorescent Stx1 B Subunit-Stx1 derived from E. coli O157:H7 was purified previously (16). The pEF-Stx1 B (binding) subunit-His 6 vector for recombinant histidine-tagged Stx1 B subunit (1BH) was constructed previously (16).…”

Monovalent Gb3-/Gb2-Derivatives Conjugated with a Phosphatidyl Residue: A Novel Class of Shiga Toxin-Neutralizing Agent

“…26,27) Moreover, the clustering of Gb 3 in lipid rafts on the cell membrane has been shown to play an important role in the binding of Stxs. 11,12) We 9) and others 21) reported on the importance of Gb 3 density per molecule for the neutralization of Stxs, especially Stx-2, using Gb 3 -copolymers with different molar ratios of sugar unit to acrylamide. Here, Gb 3 -PEDP and Gb 2 -PEDP neutralized not only Stx-1 but also Stx-2, although these compounds have a monomeric chemical structure, while both Gb 3 -fluorescein and Gb 2 -fluorescein, which are also monomer did not neutralize Stx activity, even at the higher concentration of 1 mM.…”

“…12) In light of these findings, many synthetic compounds mimicking the natural receptors have been investigated for eliminating Stxs from the intestine and/or neutralizing Stxs in the circulation, as a therapeutic strategy for protecting patients from serious Stx-mediated diseases. 9,10,[13][14][15][16][17][18][19][20][21][22] All of these previously reported compounds possess repeated Gb 3 mimic structures in a molecule with various backbones on expecting clustering effect of the saccharide moiety, although there was one exception of adamantyl-Gb 3 , 10) a Gb 3 monovalent derivative, that was speculated to assemble into small aggregates for activity.In the present study, we synthesized novel Gb 3 -and Gb 2 -conjugated derivatives with a phosphatidyl residue, a sugar unit monomer in the chemical structure, which would be expected to form liposomes and clusters of the sugar unit to neutralize Stx-1 and Stx-2. These compounds showed strong neutralizing activity against not only Stx-1 but also Stx-2.…”

Histidine-Tagged Shiga Toxin B Subunit Binding Assay: Simple and Specific Determination of Gb3 Content in Mammalian Cells

“…We used 1B-His for the detection of Gb3 because a higher affinity of 1B-His toward Gb3 was observed in comparison with 2B-His. 15) The His-tagging strategy is useful for the purification of recombinant proteins 24) and we tried to apply for their detection in our present study. HisProbe-HRP is a unique product for the detection of His-tagged protein.…”