An Orally Applicable Shiga Toxin Neutralizer Functions in the Intestine To Inhibit the Intracellular Transport of the Toxin

Abstract

Shiga toxin 2 (Stx2) is a major virulence factor in infections with Stx-producing Escherichia coli (STEC), which causes gastrointestinal diseases and sometimes fatal systemic complications. Recently, we developed an oral Stx2 inhibitor known as Ac-PPP-tet that exhibits remarkable therapeutic potency in an STEC infection model. However, the precise mechanism underlying the in vivo therapeutic effects of Ac-PPP-tet is unknown. Here, we found that Ac-PPP-tet completely inhibited fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. Interestingly, Ac-PPP-tet accumulated in the ileal epithelial cells only through its formation of a complex with Stx2. The formation of Ac-PPP-tet-Stx2 complexes in cultured epithelial cells blocked the intracellular transport of Stx2 from the Golgi apparatus to the endoplasmic reticulum, a process that is essential for Stx2 cytotoxicity. Thus, Ac-PPP-tet is the first Stx neutralizer that functions in the intestine by altering the intracellular transport of Stx2 in epithelial cells. Infection with Shiga toxin (Stx)-producing Escherichia coli(STEC) in humans causes gastrointestinal diseases that are often followed by potentially fatal systemic complications such as acute encephalopathy and hemolytic-uremic syndrome (12,22,25,26). Stx is produced in the gut, traverses the epithelium, and passes into the circulation. Circulating Stx then causes vascular damage in specific target tissues such as the brain and the kidney, resulting in systemic complications. For this reason, development of a neutralizer that specifically binds to and inhibits Stx in the gut and/or in the circulation would be a promising therapeutic approach.Stx is classified into two subgroups, Stx1 and Stx2. Stx2 is more closely related to the severity of STEC infections than Stx1 (6,23,31,33). Stx consists of a catalytic A subunit and a pentameric B subunit. The former has 28S rRNA N-glycosidase activity and inhibits eukaryotic protein synthesis, while the latter is responsible for binding to the functional cell surface receptor Gb3 [Gal␣(1-4)-Gal␤(1-4)-Glc␤1-ceramide] (11,17,25). The crystal structure of Stx reveals the presence of three distinctive binding sites (i.e., sites 1, 2, and 3) on each B subunit monomer for the trisaccharide moiety of Gb3 (7, 16). Highly selective and potent binding of Stx to Gb3 is attributed mainly to the multivalent interaction between the B subunit pentamer and the trisaccharide. This so-called clustering effect has formed the basis for the development of several synthetic Stx neutralizers that contain the trisaccharide in multiple con-

Recent Advances in Understanding Enteric Pathogenic Escherichia coli

“…Similarly, phase 1 clinical trials of chimeric monoclonal Stx1-and Stx2-neutralizing antibodies were also shown to be generally well tolerated in injected volunteers (383,384). Alternatively, short peptides may block Stx2 transport through epithelial cells (385). These cell-permeative peptides were also protective in a baboon model following Stx2 injection, as renal injury was abated (386).…”

The Crystal Structure of Shiga Toxin Type 2 with Bound Disaccharide Guides the Design of a Heterobifunctional Toxin Inhibitor

“…To be successful such approaches have to overcome the millimolar intrinsic affinity for Gb 3 analogues and, at least for oligosaccharide based antagonists, the short half-life of such polar molecules in circulation. Several interesting approaches and ligand designs have been reported (22)(23)(24)(25)(26)(27)(28)(29).…”

Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition

“…Accordingly, several compounds with clustered trisaccharides that can bind to the B subunit with high affinity have been developed and shown to effectively neutralize Stx both in vitro and in vivo (27)(28)(29)(30)(31)(32)(33). These compounds, however, cannot be customized to specific Stx subtypes, because all Stx subtypes recognize the trisaccharide as the natural binding unit.Previously, we developed a library of multivalent peptides exhibiting the clustering effect, from which we identified Stx-neutralizing tetravalent peptides by screening the library for highaffinity binding to the specific receptor-binding sites (33)(34)(35)(36). By targeting Stx2a receptor-binding site 3 or Stx1a site 1, we identified various tetravalent peptides demonstrating remarkable therapeutic potency in both a mouse model of EHEC infection (34, 36) and a nonhuman primate model (19).…”

“…Previously, we developed a library of multivalent peptides exhibiting the clustering effect, from which we identified Stx-neutralizing tetravalent peptides by screening the library for highaffinity binding to the specific receptor-binding sites (33)(34)(35)(36). By targeting Stx2a receptor-binding site 3 or Stx1a site 1, we identified various tetravalent peptides demonstrating remarkable therapeutic potency in both a mouse model of EHEC infection (34, 36) and a nonhuman primate model (19).…”