Identification of a Wide Range of Motifs Inhibitory to Shiga Toxin by Affinity-Driven Screening of Customized Divalent Peptides Synthesized on a Membrane

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli, binds to target cells through a multivalent interaction between its B-subunit pentamer and the cell surface receptor globotriaosylceramide, resulting in a remarkable increase in its binding affinity. This phenomenon is referred to as the "clustering effect." Previously, we developed a multivalent peptide library that can exert the clustering effect and identified Stx neutralizers with tetravalent peptides by screening this library for high-affinity binding to the specific receptor-binding site of the B subunit. However, this technique yielded only a limited number of binding motifs, with some redundancy in amino acid selectivity. In this study, we established a novel technique to synthesize up to 384 divalent peptides whose structures were customized to exert the clustering effect on the B subunit on a single cellulose membrane. By targeting Stx1a, a major Stx subtype, the customized divalent peptides were screened to identify high-affinity binding motifs. The sequences of the peptides were designed based on information obtained from the multivalent peptide library technique. A total of 64 candidate motifs were successfully identified, and 11 of these were selected to synthesize tetravalent forms of the peptides. All of the synthesized tetravalent peptides bound to the B subunit with high affinities and effectively inhibited the cytotoxicity of Stx1a in Vero cells. Thus, the combination of the two techniques results in greatly improved efficiency in identifying biologically active neutralizers of Stx.

志贺毒素（Stx）是肠出血性大肠杆菌的一种主要毒力因子，它通过其B亚基五聚体与细胞表面受体球三糖神经酰胺之间的多价相互作用与靶细胞结合，导致其结合亲和力显著增加。这种现象被称为“聚类效应”。此前，我们开发了一个能够产生聚类效应的多价肽库，并通过筛选该库以寻找与B亚基的特定受体结合位点的高亲和力结合，用四价肽鉴定出了Stx中和剂。然而，该技术仅产生了数量有限的结合基序，在氨基酸选择性上存在一些冗余。在本研究中，我们建立了一种新技术，可在单个纤维素膜上合成多达384种二价肽，其结构经过定制以对B亚基产生聚类效应。通过以主要的Stx亚型Stx1a为靶点，对定制的二价肽进行筛选以鉴定高亲和力结合基序。肽的序列是根据从多价肽库技术获得的信息设计的。总共成功鉴定出64个候选基序，并从中选择了11个来合成肽的四价形式。所有合成的四价肽都以高亲和力与B亚基结合，并有效抑制了Stx1a在非洲绿猴肾细胞（Vero细胞）中的细胞毒性。因此，这两种技术的结合极大地提高了鉴定Stx具有生物活性中和剂的效率。