RTX cytotoxins recognize β2 integrin receptors through N-linked oligosaccharides

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (Hly) (CyaA, ACT, or AC-Hly) is a cytotoxin of the RTX (repeat in toxin) family. It delivers into target cells an AC domain that catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting phagocyte functions. CyaA utilizes a heavily N-glycosylated beta 2 integrin receptor CD11b/CD18 (alpha(M)beta(2), Mac-1, or CR3). We show that deglycosylation of cell surface proteins by glycosiclase treatment, or inhibition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CID11b-expressing cells. Furthermore, binding of CyaA to cells was strongly inhibited in the presence of free saccharides occurring as building units of integrin oligosaccharicle complex, whereas saccharides absent from integrin oligosaccharicle chains failed to inhibit CyaA binding to CD11b/CD18-expressing cells. CyaA, hence, selectively recognized sugar residues of N-linked oligosaccharides of integrins. Moreover, glycosylation of CD11a/CD18, another receptor of the beta 2 integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA) and the Escherichia coli alpha-Hly (HlyA). These results show that binding and killing of target cells by CyaA, LtxA, and HlyA depends on recognition of N-linked oligosaccharicle chains of beta 2 integrin receptors. This sets a new paradigm for action of RTX cytotoxins.