Quorum sensing is a density-dependent form of communication in bacteria

Targeted down-modulation of monocytes and macrophages is a promising strategy for controlling excessive inflammatory processes. Bacterial protein toxins are attractive pharmacological modulation tools for this purpose because of their potency and specificity. Here, we combined the specific properties of the two protein toxins C3lim from Clostridium limosum and CyaA from Bordetella pertussis in the preferentially monocyte/macrophage-targeting bioconjugate C2IN-C3lim_E174Q-Cys_His-AC. The enzymatically inactive and monocytic cell-targeting transporter C2IN-C3lim_E174Q-Cys was recombinantly produced and chemically coupled to the isolated adenylate cyclase (AC) domain of CyaA, which mediates down-modulation of monocytic cell functions through intracellular cAMP accumulation Within 1 h, the bioconjugate showed preferential cell association with monocytic cells relative to HeLa cells, which served as the non-target cell model, without impairing overall cellular viability. Intracellular enzymatic activity was successfully confirmed by significantly elevated cAMP levels in all target cell types. Moreover, in migration assays with primary human monocytes, the bioconjugate markedly reduced both general motility and targeted chemotaxis, demonstrating that the delivery of the enzyme moiety translates into a measurable functional response. Therefore, we conclude that the novel bioconjugate C2IN-C3lim_E174Q-Cys_His-AC enables preferential targeting of monocytes and macrophages under the experimental conditions investigated here and provides proof-of-concept for a targeted protein delivery platform for functional modulation of monocytic cells.