| 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 [Galα(1-4)-Galβ(1-4)-Glcβ1-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 configurations (3, 5, 14, 18, 19, 36). These neutralizers can strongly bind to Stx and inhibit its cytotoxic activity. Some are also effective in STEC infection models (18, 19, 36). However, the clinical application of these neutralizers has been substantially hampered by the synthetic complexity of the trisaccharide moiety.We have recently screened a library of novel tetravalent peptides that exert a clustering effect and have identified four peptide motifs that are superior to trisaccharide in binding Stx (20). Tetravalent forms of these peptides bind with high affinity to one trisaccharide-binding site (site 3) of Stx2 and effectively inhibit Stx2 cytotoxicity. This is particularly true of the neutralizer designated PPP-tet, which contains four Pro-Pro-Pro-Arg-Arg-Arg-Arg motifs. PPP-tet protects mice from a fatal dose of E. coli O157:H7, even when the peptide is orally administered after the establishment of infection (20). Moreover, the addition of acetyl groups to all the amino termini of PPP-tet (yielding Ac-PPP-tet) makes this compound resistant to proteolysis and markedly enhances its protective activity against STEC infection, indicating that Ac-PPP-tet holds promise as a therapeutic agent for STEC infections.After binding to Gb3, Stx is first transported to the Golgi apparatus in a retrograde manner and then transported to the endoplasmic reticulum (ER). On the other hand, the Stx catalytic A subunit is released into the cytoplasm, where it inhibits protein synthesis (27, 29). The retrograde transport of Stx is known to be essential for Stx cytotoxicity (2, 27, 28). In Vero cells, one of the cell types most sensitive to Stx, PPP-tet prevents Stx2 cytotoxicity by inducing the aberrant transport of Stx from the Golgi apparatus to an acidic compartment rather than to the ER, leading to the degradation of Stx (20). An advantage of PPP-tet is its ability to partially permeate cells, which allows it to inhibit the cytotoxicity of Stx2 already incorporated into cells (20). Nevertheless, the precise mechanism by which PPP-tet and Ac-PPP-tet function in vivo, as well as the identities of the organs or cells targeted by these compounds, is unknown.To understand how orally administered Ac-PPP-tet functions in vivo, we investigated the effect of Ac-PPP-tet on fluid accumulation in the rabbit ileum caused by the direct injection of Stx2. The rabbit ileal loop system is highly valid for evaluating the toxicity of Stx2 produced in the intestine after infection. We also examined the localization of the tetravalent peptide and Stx2 in the intact rabbit ileum, cultured ileal specimens, and Caco-2 intestinal epithelial cells. Our results reveal that Ac-PPP-tet functions as a potent Stx2 neutralizer in the intestine by altering the intracellular transport of Stx2 in epithelial cells. |
