Previously, we demonstrated that the similar to 130-kDa CyaA-hemolysin (CyaA-Hly, Met(482)-Arg(1706)) from Bordetella pertussis was palmitoylated at Lys(983) 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, Met(482)-Leu(750)) for toxin acylation was performed. The -100-kDa RTX (Repeat-in-ToXin) fragment (CyaA-RTX, Ala(751)-Arg(1706)) containing the Ly(s983)-acylation region (AR, Ala(751)-Gln(1000)), 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 similar to 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 alpha 1-alpha 5 (Leu(500)-Val(698)) 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 Lys(983). (C) 2018 Elsevier Inc. All rights reserved.