Carbohydrate-binding specificity of the Escherichia coli cytolethal distending toxin CdtA-II and CdtC-II subunits

Intoxication by cytolethal distending toxin depends on assembly of CdtB, the active A component of this AB toxin, with the cell surface-binding (13) 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-HEd and CdtC-IIEc and their capacity to provide a binding platform for CdtBII(Ec) 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-II,, and CdtC-IIEc to these glycoproteins. In addition, removal of N- but not O-linked surface sugar attachments prevents CDT-HEc 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.