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

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.

Lysosomal swelling and lysis mediate delivery of C3 toxins to their cytoplasmic targets

“…Note that these times refer to the (chase) time elapsed after an initial 15 min (pulse) period when the toxin was allowed to bind to the plasma membrane before washing. These findings are therefore roughly consistent with those recently reported by Fellermann et al, who found that 70-100% of C3 toxin from Clostridium botulinum is associated with early endosomes of human macrophages after 30 min (Fellermann et al, 2022). Accordingly, at this stage C3bot displays a punctate pattern that resembles that of C3larvinA at a comparable total time.…”

The Clostridium botulinum C2 Toxin Subunit C2IIa Delivers Enzymes with Positively Charged N-Termini into the Cytosol of Target Cells

“…STED-microscopy was performed as described in [ 61 , 62 ]. A total of 1.8 × 10 5 HeLa cells were seeded per well in an 8-well µ-slide with glass bottom (ibidi GmbH, Gräfelfing, Germany).…”

“…For better visualization, a σ = 1 pixel Gaussian blur and >20 count intensity threshold were applied. A self-written search algorithm (Python 3.7, available on request) was used to count the eGFP signals, as described earlier [ 61 ], and a threshold of 10 counts was applied. For each experiment, the mean number of eGFP signals was calculated and averaged for five individual experiments ( n = 5).…”

Lysozyme: an endogenous antimicrobial protein with potent activity against extracellular, but not intracellular Mycobacterium tuberculosis

“…Furthermore, the shortening of lysozyme to Lys-H1 may affect and limit its antimycobacterial activity. The uptake and trafficking of lysozyme or its derivatives could be improved by engineering delivery vehicles that specifically target macrophages [12] and support the migration of effector molecules into the mycobacterial compartment. The efficacy of this approach has been recently demonstrated for the endogenous antimycobacterial peptides gran1…”