[en] Highlights: • Acylation of CyaA-Hly by CyaC-acyltransferase requires AR-acylation and HP-hydrophobic regions, hence hemolysis activation. • ∼100-kDa AR-containing CyaA-RTX lacking HP cannot be acylated by functional CyaC, thus hemolytically inactive. • ∼70-kDa hemolytically inactive-CyaA-HP/BI containing both AR and HP could interact with co-expressed CyaC. • ∼20-kDa CyaA-HP comprising α1-α5 potentially interacted with CyaC via multiple H-bonding and ionic interactions. • HP is required for not only membrane-pore formation but also CyaC-association, hence toxin acylation. Previously, we demonstrated that the ∼130-kDa CyaA-hemolysin (CyaA-Hly, Met⁴⁸²-Arg¹⁷⁰⁶) from Bordetella pertussis was palmitoylated at Lys⁹⁸³ when co-expressed with CyaC-acyltransferase in Escherichia coli, and thus activated its hemolytic activity. Here, further investigation on a possible requirement of the N-terminal hydrophobic region (HP, Met⁴⁸²-Leu⁷⁵⁰) for toxin acylation was performed. The ∼100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala⁷⁵¹-Arg¹⁷⁰⁶) containing the Lys⁹⁸³-acylation region (AR, Ala⁷⁵¹-Gln¹⁰⁰⁰), but lacking HP, was co-produced with CyaC in E. coli. Hemolysis assay indicated that CyaA-RTX showed no hemolytic activity. Additionally, MALDI-TOF/MS and LC-MS/MS analyses confirmed that CyaA-RTX was non-acylated, although the co-expressed CyaC-acyltransferase was able to hydrolyze its chromogenic substrate−p-nitrophenyl palmitate and acylate CyaA-Hly to become hemolytically active. Unlike CyaA-RTX, the ∼70-kDa His-tagged CyaA-HP/BI fragment which is hemolytically inactive and contains both HP and AR was constantly co-eluted with CyaC during IMAC-purification as the presence of CyaC was verified by Western blotting. Such potential interactions between the two proteins were also revealed by semi-native PAGE. Moreover, structural analysis via electrostatic potential calculations and molecular docking suggested that CyaA-HP comprising α1-α5 (Leu⁵⁰⁰-Val⁶⁹⁸) can interact with CyaC through several hydrogen and ionic bonds formed between their opposite electrostatic surfaces. Overall, our results demonstrated that the HP region of CyaA-Hly is conceivably required for not only membrane-pore formation but also functional association with CyaC-acyltransferase, and hence effective palmitoylation at Lys⁹⁸³.