Uptake of Clostridium botulinum C3 Exoenzyme into  Intact HT22 and J774A.1 Cells

Rohrbeck, Astrid; Elsner, Leonie von; Hagemann, Sandra; Just, Ingo

doi:10.3390/toxins7020380

Cited by 18 publications

(31 citation statements)

References 56 publications

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“…Western blot analysis again confirmed depletion of vimentin in the synaptosomal fraction compared to the homogenate. The interaction of surface vimentin with clostridial C3 transferase was first shown by our group in neuronal and macrophage cell lines (Rohrbeck et al, 2014(Rohrbeck et al, , 2015. Indeed, incubation of spinal cord synaptosomes for 1 hr at 4 C with 8 and 40 ng/μl of recombinant protein followed by stringent washing resulted in a concentration-dependent binding of vimentin ( Figure 6b).…”

Section: Resultsmentioning

confidence: 66%

“…This might be indicative of an accelerated C3bot-uptake and ADP-ribosylation (followed by proteasomal degradation;Rohrbeck, von Elsner, Hagemann, & Just, 2015) in the presence of extracellularly added vimentin in the knockout. Incubation of astrocytes with C3bot alone resulted in the expected stellate morphology in wild-type cells but was, to a lesser extent, also observed in vimentin knockout astrocytes (Figure 2c).Addition of vimentin alone was without any effect in either genotype.The combinatory treatment with C3bot plus vimentin, however, resulted in an intensified stellation, notably enhanced in the knockout.Western blot analysis of rhoA levels showed a complete ADPribosylation of rhoA (detected by the higher molecular weight) in the presence of C3bot both in the wild-type and in the knockout (Figure 2d).…”

mentioning

confidence: 97%

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Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons

Adolf

Rohrbeck

Münster-Wandowski

et al. 2018

Glia

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Clostridium botulinum C3 transferase (C3bot) ADP‐ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild‐type with Vim−/− mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP‐ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin‐binding RGD motif (C3bot‐G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross‐talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild‐type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim−/− astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.

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Section: Resultsmentioning

confidence: 66%

mentioning

confidence: 97%

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons

Adolf

Rohrbeck

Münster-Wandowski

et al. 2018

Glia

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“…C3 was internalized even if the formation of the clathrin-coated vesicles is blocked by acidification of the cytosol (Sandvig et al 1987) or when clathrin assembly and disassembly are inhibited by chlorpromazine (Wang et al 1993). This indicates that clathrin-coated pits did not play a role in the uptake of C3 (Rohrbeck et al 2015). Also, bafilomycin A1, an inhibitor of the vacuolar proton ATPase, did not inhibit the uptake of C3.…”

Section: Classical Endocytosis Mechanism and Internalization Of C3 Exmentioning

confidence: 92%

“…Lipid rafts play an important role in uptake of numerous protein toxins. However, filipin and methyl-beta-cyclodextrin (MBCD) did not show any significant effect on uptake of C3, although MBCD was effective in inhibiting the uptake of C. difficile toxin B (Rohrbeck et al 2015). Therefore, the uptake mechanism of C3 exoenzyme seems to be independent of cholesterol.…”

Section: Classical Endocytosis Mechanism and Internalization Of C3 Exmentioning

confidence: 98%

“…Meanwhile, it is accepted that C3 enters eukaryotic cells despite the absence of a binding and translocation domain. In addition, recent findings show that C3 causes ADP-ribosylation of RhoA in a short time and at nanomolar concentrations (Ahnert-Hilger et al 2004;Fahrer et al 2010;Rohrbeck et al 2015). The development of alternative application techniques in fact prevented a detailed study of binding and uptake of C3.…”

Section: C3 Exoenzyme As Cell Biological Toolmentioning

confidence: 99%

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Cell Entry of C3 Exoenzyme from Clostridium botulinum

Rohrbeck

Just

2016

Current Topics in Microbiology and Immunology

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Clostridium botulinum C3 is the prototype of C3-like ADP-ribosyltransferases that selectively ADP-ribosylate the small GTP-binding proteins RhoA/B/C and inhibit their downstream signaling pathways. It is used as pharmacological tool to study cellular Rho functions. In addition, C3bot harbors a transferase-independent activity on neurons to promote axonal and dendritic growth and branching. Many bacterial protein toxins interact specifically with proteins and/or other membrane components at the surface of target cells. Binding enables access to the appropriate cellular compartment so that the knowledge of the receptor allows essential insight into the mechanism of these toxins. Unlike other bacterial protein toxins (such as the clostridial C1 and C2 toxins from C. botulinum), C3 exoenzyme is devoid of a binding and translocation domain, with which toxins usually initiate receptor-mediated endocytosis followed by access to the intact cell. To date, no specific mechanism for internalization of C3 exoenzyme has been identified. Recently, vimentin was identified as membranous C3-binding partner involved in binding and uptake of C3. Although vimentin is not detected in neurons, vimentin is re-expressed after damage in regenerating neurons. Reappearance of vimentin allows C3 to get access to lesioned neurons/axons to exhibit axonotrophic and dentritotrophic effects.

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Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions

Heck

Ostertag

Schnell

et al. 2019

Adv Healthcare Materials

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The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho‐GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a “mix‐and‐match” approach containing the specific Rho‐inhibiting clostridial C3 enzyme and three PMN‐binding peptides using an avidin platform. Selective delivery of the C3 Rho‐inhibitor with these complexes into the cytosol of human neutrophil‐like NB‐4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type‐selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post‐traumatic injury.

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Uptake of Clostridium botulinum C3 Exoenzyme into Intact HT22 and J774A.1 Cells

Cited by 18 publications

References 56 publications

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons

Cell Entry of C3 Exoenzyme from Clostridium botulinum

Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions

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