Recombinant streptavidin-C3bot for delivery of proteins into macrophages

Christow, Hannes; Lillich, Maren; Sold, Alexander; Fahrer, Jörg; Barth, Holger

doi:10.1016/j.toxicon.2013.02.002

Cited by 6 publications

(12 citation statements)

References 9 publications

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“…Using enzymatically inactive toxin mutants as vaccine carrier is a common method and the most prominent example is the nontoxic mutant CRM197 of DT, which is well established and administered as carrier protein worldwide [34,35]. In this regard, we already demonstrated that different cargo molecules can infiltrate into the cytosol of macrophages or monocytes via C3bot E174Q -mediated transport [55][56][57]. Such an approach might be used to initiate immune responses against cancer cells as discussed for several types of cancers [30].…”

Section: Discussionmentioning

confidence: 99%

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells

Fellermann

Huchler

Fechter

et al. 2020

Toxins

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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.

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Section: Discussionmentioning

confidence: 99%

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells

Fellermann

Huchler

Fechter

et al. 2020

Toxins

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“…J774A.1 macrophages incubated with biotin-labeled DTA (B-DTA) or with B-DTA, which was previously bound to the transporter molecule C3botE174Q-SA. When B-DTA was delivered into the cytosol by C3botE174Q-SA the cells died, while cells treated with B-DTA alone stayed viable [modified from Christow et al ( 58 )].…”

Section: Molecular and Cellular Consequences Of The C3-catalyzed Rho mentioning

confidence: 99%

“…Prompted by this first successful exploitation of C3 for protein delivery, novel modular transporters based on biotin/streptavidin technology were developed ( 58 , 59 ), as depicted in Figure 2 B. Here, the C3botE174Q moiety mediates the specific transport of streptavidin into the cytosol of monocytes/macrophages.…”

Section: Molecular and Cellular Consequences Of The C3-catalyzed Rho mentioning

confidence: 99%

“…Here, the C3botE174Q moiety mediates the specific transport of streptavidin into the cytosol of monocytes/macrophages. Streptavidin was either coupled to C3botE174Q by chemical crosslinking ( 59 ) or by genetic fusion ( 58 ) and serves as a delivery platform for biotin-labeled cargo molecules, which are then released in the host cell cytosol to elicit their pharmacological effects, as demonstrated for the enzymatic domain DTA of diphtheria toxin ( 58 ), which triggers cell death by inactivating the elongation factor EF-2, which is essential for protein biosynthesis ( 60 ) (see Figure 2 B). In conclusion, enzymatically inactive C3 protein serves as a transport system for delivery of “foreign” proteins including pharmacologically active enzymes into the cytosol of cultured monocytes/macrophages.…”

Section: Molecular and Cellular Consequences Of The C3-catalyzed Rho mentioning

confidence: 99%

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Clostridial C3 Toxins Target Monocytes/Macrophages and Modulate Their Functions

et al. 2015

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The C3 enzymes from Clostridium (C.) botulinum (C3bot) and Clostridium limosum (C3lim) are single chain protein toxins of about 25 kDa that mono-ADP-ribosylate Rho-A, -B, and -C in the cytosol of mammalian cells. We discovered that both C3 proteins are selectively internalized into the cytosol of monocytes and macrophages by an endocytotic mechanism, comparable to bacterial AB-type toxins, while they are not efficiently taken up into the cytosol of other cell types including epithelial cells and fibroblasts. C3-treatment results in disturbed macrophage functions, such as migration and phagocytosis, suggesting a novel function of clostridial C3 toxins as virulence factors, which selectively interfere with these immune cells. Moreover, enzymatic inactive C3 protein serves as a transport system to selectively deliver pharmacologically active molecules into the cytosol of monocytes/macrophages without damaging these cells. This review addresses also the generation of C3-based molecular tools for experimental macrophage pharmacology and cell biology as well as the exploitation of C3 for development of novel therapeutic strategies against monocyte/macrophage-associated diseases.

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“…Recently we and others identified monocytes/macrophages as target cells for the C3 protein toxins (~25 kDa) from Clostridium ( C. ) botulinum (C3bot1) and C. limosum (C3lim) (Fahrer et al 2010 ; Dmochewitz et al 2013 ; Christow et al 2013 ; Rotsch et al 2012 ; Tautzenberger et al 2013 ; Rohrbeck et al 2014 ). The clostridial C3 toxins catalyze the covalent transfer of an ADP-ribose moiety from NAD onto Asn-41 of the GTPases Rho A, -B, -C (Aktories and Frevert 1987 ; Aktories et al 1995 ; Just et al 1992 ; Han et al 2001 ; for review see Aktories 2011 ), which inhibits the Rho-mediated signal transduction in mammalian cells (Rubin et al 1988 ; Chardin et al 1989 ; Etienne-Manneville and Hall 2002 ).…”

Section: Introductionmentioning

confidence: 99%

Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo

et al. 2017

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Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-017-2058-y) contains supplementary material, which is available to authorized users.

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Recombinant streptavidin-C3bot for delivery of proteins into macrophages

Cited by 6 publications

References 9 publications

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells

Clostridial C3 Toxins Target Monocytes/Macrophages and Modulate Their Functions

Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo

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