title: 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: 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 subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC.
authors.affiliation: Department of Molecular Life Sciences, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Mitsui
authors.affiliation: Department of Molecular Life Sciences, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Watanabe-Takahashi
authors.affiliation: Department of Molecular Life Sciences, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Eiko
authors.initials: E
authors.lastname: Shimizu
authors.affiliation: Department of Molecular Life Sciences, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Baihao
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Funamoto
authors.affiliation: International Prevention of Epidemics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Yamasaki
authors.affiliation: Department of Molecular Life Sciences, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan knishika@mail.doshisha.ac.jp.
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Nishikawa
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D054324:Enterohemorrhagic Escherichia coli / Q000378:metabolism; D004927:Escherichia coli Infections / Q000378:metabolism / Q000382:microbiology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D022641:Shiga Toxin 2 / Q000378:metabolism*; D014709:Vero Cells; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D019151:Peptide Library; D010455:Peptides; D022641:Shiga Toxin 2; D037521:Virulence Factors