*Article Clostridium botulinum* **C3 Toxin for Selective Delivery of Cargo into Dendritic Cells and Macrophages**

**Maximilian Fellermann 1, Mia Stemmer 1, Reiner Noschka 2, Fanny Wondany 3, Stephan Fischer 1, Jens Michaelis 3, Steffen Stenger <sup>2</sup> and Holger Barth 1,\***


**Abstract:** The protein toxin C3bot from *Clostridium botulinum* 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 C3botE174Q as transporters for selective delivery of cargo molecules into macrophages and DCs. C3bot and C3botE174Q 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 C3botE174Q strongly enhanced the cytosolic release of functional eGFP. Subsequently, a modular system was created on the basis of C3botE174Q 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 C3botE174Q is a promising candidate for the cell-type-selective delivery of small molecules, peptides, and proteins into the cytosol of macrophages and DCs.

**Keywords:** clostridial C3 toxin; *Clostridium botulinum*; C3bot; C3botE174Q; dendritic cells; macrophages; monocytes; stimulated emission depletion (STED); super-resolution microscopy

**Key Contribution:** C3bot and especially the non-toxic variant C3botE174Q can be used as cell-typeselective molecular Trojan horses for transporting different cargo molecules into the cytosol of macrophages and dendritic cells.
