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Infection and Immunity, December 2006, p. 6992-6998, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.01247-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Michael J. Smith, Humberto M. Carvalho, Angela R. Melton-Celsa, and Alison D. O'Brien*

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Room B4052, 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799

Received 4 August 2006/ Returned for modification 8 September 2006/ Accepted 28 September 2006

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Room B4052, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Phone: (301) 295-3400. Fax: (301) 295-3773. E-mail: aobrien{at}usuhs.mil.

{triangledown} Published ahead of print on 9 October 2006.

Editor: D. L. Burns

Infection and Immunity, December 2006, p. 6992-6998, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.01247-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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