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Infection and Immunity, April 2005, p. 2051-2060, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2051-2060.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Carbohydrate-Binding Specificity of the Escherichia coli Cytolethal Distending Toxin CdtA-II and CdtC-II Subunits

Leslie A. McSweeney and Lawrence A. Dreyfus*

Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri—Kansas City, Kansas City, Missouri

Received 22 July 2004/ Returned for modification 24 September 2004/ Accepted 23 November 2004

Intoxication by cytolethal distending toxin depends on assembly of CdtB, the active A component of this AB toxin, with the cell surface-binding (B) component, composed of the CdtA-CdtC heterodimer, to form the active holotoxin. Here we examine the cell surface binding properties of Escherichia coli-derived CdtA-II (CdtA-IIEc) and CdtC-IIEc and their capacity to provide a binding platform for CdtB-IIEc. Using a flow cytometry-based binding assay, we demonstrate that CdtB-IIEc binds to the HeLa cell surface in a CdtA-IIEc- and CdtC-IIEc-dependent manner and that CdtA-IIEc and CdtC-IIEc compete for the same structure on the HeLa cell surface. Preincubation of cells with glycoproteins (thyroglobulin and fetuin), but not simple sugars, blocks surface binding of CdtA-IIEc and CdtC-IIEc. Moreover, CdtA-IIEc and CdtC-IIEc bind immobilized fetuin and thyroglobulin as well as fucose and to a lesser degree N-acetylgalactoseamine and N-acetylglucoseamine. Removal of N- but not O-linked carbohydrates from fetuin and thyroglobulin prevents binding of CdtA-IIEc and CdtC-IIEc to these glycoproteins. In addition, removal of N- but not O-linked surface sugar attachments prevents CDT-IIEc intoxication. To characterize the cell surface ligand recognized by CdtA-IIEc and CdtC-IIEc, lectins having various carbohydrate specificities were used to block CDT activity and the cell surface binding of CdtA-IIEc and CdtC-IIEc. Pretreatment of cells with AAA, SNA-I, STA, UEA-I, GNA, and NPA partially or completely blocked CDT activity. AAA, EEA, and UEA-I lectins, all having specificity for fucose, blocked surface binding of CdtA-IIEc and CdtC-IIEc. Together, our data indicate that CdtA-IIEc and CdtC-IIEc bind an N-linked fucose-containing structure on HeLa cells.

* Corresponding author. Mailing address: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri—Kansas City, 5007 Rockhill Rd., Kansas City, MO 64110. Phone: (816) 235-2590. Fax: (816) 235-5158. E-mail: dreyfusl{at}umkc.edu.

Editor: V. J. DiRita

Infection and Immunity, April 2005, p. 2051-2060, Vol. 73, No. 4
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.4.2051-2060.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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