Updated publication reference for PubMed record(s): 32393579. The Bordetella adenylate cyclase toxinhemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli both belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLβ2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and permeabilize also a variety of other cells. CyaA bears an Nterminal adenylyl cyclase enzyme (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety and binds the complement receptor 3 (CR3, αMβ2, CD11b/CD18 or Mac-1) of myeloid phagocytes, penetrates their plasma membrane and delivers into cytosol the AC enzyme. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the much shorter HlyCacylated and LFA-1-binding RTX moiety of HlyA. Instead of binding CR3, a CyaA1710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered the AC enzyme into Jurkat lymphoblastoma T cells. At high chimera concentrations (25 nM), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated RTX moiety of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results allow to delimit the residues 400 to 710 of CyaA as the 'AC translocon' sufficient for translocation of the AC enzyme polypeptide across the plasma membrane of target cells