<i>Clostridium botulinum</i> C3 Toxin for Selective Delivery of Cargo into Dendritic Cells and Macrophages.

The protein toxin C3bot from <i>Clostridium botulinum</i> is a mono-ADP-ribosyltransferase that selectively intoxicates monocyte-derived cells such as macrophages, osteoclasts, and dendritic cells (DCs) by cytosolic modification of Rho-A, -B, and -C. Here, we investigated the application of C3bot as well as its non-toxic variant C3bot_E174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3bot_E174Q facilitated the uptake of eGFP into early endosomes of human-monocyte-derived macrophages, as revealed by stimulated emission depletion (STED) super-resolution microscopy. The fusion of the cargo model peptide eGFP neither affected the cell-type selectivity (enhanced uptake into human macrophages ex vivo compared to lymphocytes) nor the cytosolic release of C3bot. Moreover, by cell fractionation, we demonstrated that C3bot and C3bot_E174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3bot_E174Q for covalent linkage of cargos via thiol-maleimide click chemistry. The functionality of this system was proven by loading small molecule fluorophores or an established reporter enzyme and investigating the cellular uptake and cytosolic release of cargo. Taken together, non-toxic C3bot_E174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.