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

Abstract

c Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtypeselective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), which causes bloody diarrhea, hemorrhagic colitis, and sometimes life-threatening systemic complications such as acute encephalopathy and hemolytic-uremic syndrome (HUS) (1-6). To date, numerous EHEC strains that produce various Stx subtypes have been reported (7,8). These Stxs can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes, such as Stx1a,. Stx2a (10, 11) and Stx2d, which is activated by elastase derived from the intestinal mucosa (12-14), are highly virulent and have been linked with HUS, the most serious sequela of EHEC infection. The pathophysiologic importance of these subtypes was also confirmed by the finding that Stx2a and Stx2d are highly toxic when injected into mice (15, 16) or primates (17-19). Therefore, Stx neutralizers, particularly those customized to specifically neutralize Stx2a and Stx2d, would be highly valuable therapeutic agents for treating infections caused by various EHEC strains.Stx molecules consist of a catalytic A subunit, which has RNA N-glycosidase activity and inhibits eukaryotic protein synthesis (20,21), and a B-subunit pentamer. The B-subunit pentamer is responsible for high-affinity binding to the functional cell surface receptor Gb3 (Gal␣[1-4]-Gal␤[1-4]-Glc␤-ceramide) (4,22,23) or Gb4 (GalNAc␤[1-3]-Gal␣[1-4]-G...

Identification of a peptide motif that potently inhibits two functionally distinct subunits of Shiga toxin

“…Screening of the library led to the identification of tetravalent peptides that bind to the B-subunit pentamer and inhibit its toxicity in vitro and in vivo 20 – 22 . One of these peptides, MMA-tet (containing the following motif, Met-Met-Ala-Arg-Arg-Arg-Arg), inhibits the major Stx family members (e.g., Stx1a and Stx2a), as well as other highly virulent Stx2 subtypes, including Stx2d and Stx2c 23 .…”

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection

“…Screening of tetravalent peptides synthesized on a membrane. Spot synthesis of peptides on a cellulose membrane was performed using a ResPep SL SPOT synthesizer (INTAVIS Bioanalytical Instruments AG, Koeln, Germany) 18,19 . Fmocβ-Ala-OH (Watanabe Chemical Industries, Japan) was used in the first cycle, followed by amino hexanoic acid as a spacer.…”

“…Furthermore, we established a technique to use information obtained from the multivalent peptide library screen to synthesize hundreds of potentially target-specific tetravalent peptides on a cellulose membrane 18 . By screening the membrane, we identified a series of selective Stx neutralizers that can distinguish among closely related Stx subtypes 19 . Based on those results, we hypothesized that the combination of the multivalent peptide library screen and the cellulose membrane-based synthesis of candidate peptides could be used to identify highly specific neutralizers against IAV HA.…”

Experimental In Vivo Models of Bacterial Shiga Toxin-Associated Hemolytic Uremic Syndrome

“…Instead of vaccine development, toxin-neutralizing therapeutics using several anti-Stxs antibodies were tested in animal models like piglets or rodents, and these antibody treatments effectively rescued the Stx-intoxicated animals from severe mortality [35,[106][107][108]. In addition, peptide-based neutralizer that directly binds to Stx2 was identified to inhibit Stx binding to Gb3 receptor and successfully protected rodent models from Stx-caused lethality [109][110][111]. As therapeutic targeting for retrograde trafficking to the Golgi apparatus or the ER of the Stxs, small molecule compounds such as Retro-1, Retro-2 and Exo2 were developed and found to be protective for STECinfected mice from Stx-induced toxicity [112,113].…”