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 (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-II(Ec)) and CdtC-II(Ec) and their capacity to provide a binding platform for CdtB-II(Ec). Using a flow cytometry-based binding assay, we demonstrate that CdtB-II(Ec) binds to the HeLa cell surface in a CdtA-II(Ec)- and CdtC-II(Ec)-dependent manner and that CdtA-II(Ec) and CdtC-II(Ec) 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-II(Ec) and CdtC-II(Ec). Moreover, CdtA-II(Ec) and CdtC-II(Ec) 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(Ec) and CdtC-II(Ec) to these glycoproteins. In addition, removal of N- but not O-linked surface sugar attachments prevents CDT-II(Ec) intoxication. To characterize the cell surface ligand recognized by CdtA-II(Ec) and CdtC-II(Ec), lectins having various carbohydrate specificities were used to block CDT activity and the cell surface binding of CdtA-II(Ec) and CdtC-II(Ec). 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-II(Ec) and CdtC-II(Ec). Together, our data indicate that CdtA-II(Ec) and CdtC-II(Ec) bind an N-linked fucose-containing structure on HeLa cells.