The 13C4 Monoclonal Antibody That Neutralizes Shiga Toxin Type 1 (Stx1) Recognizes Three Regions on the Stx1 B Subunit and Prevents Stx1 from Binding to Its Eukaryotic Receptor Globotriaosylceramide

Abstract

The 13C4 monoclonal antibody (MAb) recognizes the B subunit of Stx1 (StxB1) and neutralizes the cytotoxic and lethal activities of Stx1. However, this MAb does not bind to the B polypeptide of Stx2, despite the 73% amino acid sequence similarity between StxB1 and StxB2. When we compared the amino acid sequences of StxB1 and StxB2, we noted three regions of dissimilarity (amino acids 1 to 6, 25 to 32, and 54 to 61) located near each other on the crystal structure of StxB1. To identify the 13C4 epitope, we generated seven Stx1/Stx2 B chimeric polypeptides that contained one, two, or three of the dissimilar StxB1 regions. The 13C4 MAb reacted strongly with StxB1 and the triple-chimeric B subunit but not with the other chimeras. Mice immunized with the triple-chimeric B subunit survived a lethal challenge with Stx1 but not Stx2, substantiating the identified regions as the 13C4 MAb epitope and suggesting that the incorporation of this epitope into StxB2 altered sites necessary for anti-Stx2-neutralizing Ab production. Next, single amino acid substitutions were made in StxB1 to mimic Stx1d, a variant not recognized by the 13C4 MAb. The 13C4 MAb reacted strongly to StxB1 with the T1A or G25A mutations but not with the N55T change. Finally, we found that the 13C4 MAb blocked the binding of Stx1 to its receptor, globotriaosyl ceramide. Taken together, these results indicate that the 13C4 MAb prevents the interaction of Stx1 with its receptor by binding three nonlinear regions of the molecule that span receptor recognition sites on StxB1, one of which includes the essential residue 55N.In the United States, Shiga toxin-producing Escherichia coli (STEC) strains account for about 110,000 infections per year (16). Enterohemorrhagic E. coli (EHEC) strains are a subset of STEC that contain a large pathogenicity island called the locus of enterocyte effacement and carry a 90-kb plasmid (13,30,32). Enterohemorrhagic E. coli serotype O157:H7 in particular is noted for it association with food-, water-, or petting zoo-linked outbreaks of Stx-mediated disease (4,23,28). A possible complication from an infection with an Stx-producing organism is the hemolytic uremic syndrome (HUS), a syndrome that is characterized by hemolytic anemia, thrombic thrombocytopenia, and renal failure. The majority of patients with HUS are children (5, 23), and there is a 5 to 10% fatality rate for those individuals. Furthermore, HUS survivors may have lasting kidney damage. Currently, there are no FDA-approved therapies or vaccines in the United States to combat or prevent illness from STEC, but several promising options for the future are under investigation. These options include receptor mimics and active and passive immunization strategies (reviewed in references 17 and 31). Several recent vaccine candidates not described in the above-mentioned reviews include Stx1 and Stx2 genetic toxoids, a plantbased Stx2 toxoid, and a chimeric StxA2/StxB1 toxoid that elicits a neutralizing antibody response and provides protection against a lethal challenge of b...

Monoclonal Antibody 11E10, Which Neutralizes Shiga Toxin Type 2 (Stx2), Recognizes Three Regions on the Stx2 A Subunit, Blocks the Enzymatic Action of the Toxin In Vitro, and Alters the Overall Cellular Distribution of the Toxin

“…Several explanations for this latter observation are conceivable. These possibilities include that the Western reactivity is primarily due to the interaction of 11E10 with region A ( 42 NHTPPGSY 49 ) or that partial refolding of the A subunit occurs during the Western blot process, as we observed for MAb 13C4 in another study (26).…”

“…Purified Stx1, Stx2, or sonic lysates of bacteria that expressed chimeric Stx1/Stx2 toxins were subjected to SDS-polyacrylamide gel (26). The concentrations of the A subunits in sonic lysates that contained Stx1, Stx2, or the chimeric toxins were estimated as follows.…”

“…In vitro neutralization assays of sonic lysates from bacteria that contained Stx1, Stx2, the chimeric Stx1/Stx2 toxins, Stx2c, Stx2d, Stx2d act , or Stx2e were carried out with 11E10 on Vero cells (ATCC, Manassas, VA) as described previously (14,26). MAb 11E10 neutralized Stx2 such that only 35% of the wild-type toxic activity remained.…”

“…The Stx1 ϩABC toxoid was purified with a nickel affinity column as previously described (26). The concentration of the toxoid was determined by bicinchoninic acid assay (Pierce, Rockford, IL).…”

Isolation and Identification of an Enterobacter cloacae Strain Producing a Novel Subtype of Shiga Toxin Type 1

“…The amino acid substitutions in binding sites 1 and 2 of Stx1e may also explain its lack of recognition by the Stx1-neutralizing monoclonal antibody 13C4. The epitope recognized by 13C4 has been mapped to a nonlinear region overlapping binding sites 1 and 2 of StxB1 (33). An asparagine at position 55 of StxB1 was found to be essential for 13C4 recognition.…”