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.

FIG 1

Identification of Asn16-dependent 2dBH binding motifs by screening of tetravalent peptides synthesized on a cellulose membrane. (A) General structure of the tetravalent peptides synthesized on a cellulose membrane as described previously (37). The density of the tetravalent peptide was maximized by using Fmoc-β-Ala-OH without butoxycarbonyl-β-Ala-OH for the first peptide synthesis cycle. After the addition of one aminohexanoic acid (U) as a spacer following the first β-Ala, Fmoc-Lys(Fmoc)-OH was used for the next two cycles to form four branches in the peptide chain for subsequent synthesis of the various motifs examined in this study (R = Met-Ala-[indicated motif]-Ala-). (B) The tetravalent form of the XMA-RRRR or MMX-RRRR (X indicates any amino acid except Cys) motif was synthesized on a membrane (left and center). The first three amino acids present in the motif are indicated on the right. The membrane was blotted with ¹²⁵I-2dBH or ¹²⁵I-2dBH-N16A (1 μg/ml), and the radioactivity bound to each peptide spot was quantified as a pixel value. (C) The sum of the pixel values of all of the peptide spots was normalized to 19 (the number of tetravalent peptides synthesized on the membrane) so that each peptide would have a value of 1 in the absence of selectivity. The ratio of ¹²⁵I-2dBH binding (2dBH-binding value) to ¹²⁵I-2dBH-N16A binding (N16A-binding value) (2dBH/N16A) was calculated, and the sum of each ratio was also normalized to 19 to evaluate the specificity of binding through Asn16. The product of the 2dBH-binding value and the normalized 2dBH/N16A ratio (2dBH × ratio) was used to evaluate both binding intensity and specificity. The sequences were sorted in descending order on the basis of the 2dBH-binding values. 2dBH-binding values of >1.20 and the highest 2dBH × ratio products are shaded.

FIG 2

Inhibition of the cytotoxicity of Stx2a and Stx2d for Vero cells by the tetravalent peptides identified in this study. The effects of the tetravalent peptides indicated on the cytotoxicity of Stx2a (10 pg/ml; A and C) or Stx2d (80 pg/ml; B and D) for Vero cells were examined with a cytotoxicity assay. Data are presented as percentages of the control value (mean ± standard error, n = 3; *, P < 0.05; **, P < 0.01 [Tukey's test, compared with MMA-tet]).

FIG 3

Analysis of the binding of LMA-tet and MMM-tet to 2aBH or 2dBH with the AlphaScreen assay. (A) The binding of biotinylated LMA-tet and MMM-tet to 2aBH, 2dBH, and their mutant forms (10 μg/ml) was examined with the AlphaScreen assay. Data are presented as signal intensity (mean number of counts per second ± standard error, n = 3). (B) The apparent K_D values of LMA-tet and MMM-tet for binding to 2aBH, 2dBH, and their mutant forms were determined as the concentration of the compound yielding half of the maximum binding value. The maximum values of LMA-tet and MMM-tet binding are presented as percentages of the values of their binding to 2aBH and 2dBH. —, not determined. *, P < 0.05; ***, P < 0.005 (Tukey's test); **, P < 0.005 (Student's t test, compared with 2aBH or 2dBH).