Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells.

Maximilian Fellermann,Thomas F E Barth,Fanny Wondany,Jens Michaelis,Kevin Mellert,Lea Fechter,Daniel Mayer,Christina Huchler,Stephan Fischer,Holger Barth,Peter Möller,Tobias Kolb,Steffen Stenger

doi:10.3390/toxins12090563

Maximilian Fellermann, Thomas F E Barth + Show 11 more

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https://doi.org/10.3390/toxins12090563

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Abstract

C3 protein toxins produced by Clostridium (C.) botulinum and C. limosum are mono-ADP-ribosyltransferases, which specifically modify the GTPases Rho A/B/C in the cytosol of monocytic cells, thereby inhibiting Rho-mediated signal transduction in monocytes, macrophages, and osteoclasts. C3 toxins are selectively taken up into the cytosol of monocytic cells by endocytosis and translocate from acidic endosomes into the cytosol. The C3-catalyzed ADP-ribosylation of Rho proteins inhibits essential functions of these immune cells, such as migration and phagocytosis. Here, we demonstrate that C3 toxins enter and intoxicate dendritic cells in a time- and concentration-dependent manner. Both immature and mature human dendritic cells efficiently internalize C3 exoenzymes. These findings could also be extended to the chimeric fusion toxin C2IN-C3lim. Moreover, stimulated emission depletion (STED) microscopy revealed the localization of the internalized C3 protein in endosomes and emphasized its potential use as a carrier to deliver foreign proteins into dendritic cells. In contrast, the enzyme C2I from the binary C. botulinum C2 toxin was not taken up into dendritic cells, indicating the specific uptake of C3 toxins. Taken together, we identified human dendritic cells as novel target cells for clostridial C3 toxins and demonstrated the specific uptake of these toxins via endosomal vesicles.

Highlights

Bacterial C3 protein toxins mono-ADP-ribosylate the small Rho-GTPases Rho A, B, and C at the amino acid residue Asn-41 [1]
TheseToxins changes were visible when the cells were treated with C2IN-C3lim and C3bot alone
Values are given as mean ± standard deviation (SD) (n = 3). (b) Cultured U-DCS cells were treated with C2IN-C3lim/C2IIa (40/66 nM), C2IN-C3lim (80 nm) or were left untreated at 37 °C. (c) Cultured U-DCS cells were treated with C2IN-C3lim/C2IIa (40/66 nM), C3bot (200 nM) or were left untreated at 37 °C. (b,c) After 5 h incubation time, cells were washed, lysed, and incubated with the respective freshly added C3 (300 ng) and biotin-NAD+ (10 μM) for 30 min at 37 °C

Summary

Introduction

Bacterial C3 protein toxins mono-ADP-ribosylate the small Rho-GTPases Rho A, B, and C at the amino acid residue Asn-41 [1]. The glutamic acid residue at position 174 (without signal sequence), which is important for the ADP-ribosylation activity, is conserved in the C3 ADP-ribosyltransferase family [1]. C3-like ADP-ribosyltransferase family [4]. Despite obvious similarities of the C3 family members to A-subunits of AB-type toxins, no corresponding B-subunits have been identified for the C3 proteins so far [1,6]. Some exceptions are C3-like toxins such as PlxA and C3larvin, where a B subunit is suspected to be responsible for specific uptake into cells [7,8,9]. Classical C3 toxins are poorly internalized into most cell types, requiring high C3 concentrations (400–4000 nM) and long incubation times (>24 h) [5,10]

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