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Vimentin Mediates Uptake of C3 Exoenzyme
Abstract and Figures
Clostridium botulinum C3 exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 exoenzyme.
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... Despite the lack of a classical cell-binding subunit of C3bot (as in the case of the clostridial neurotoxins), we were able to demonstrate that the intermediate filament protein vimentin as well as the ß1-subunit of integrins, known to serve as a transmembrane receptor for cell-extracellular matrix adhesion, function as surface interaction partners for C3bot (Rohrbeck et al., 2014(Rohrbeck et al., , 2017Adolf et al., 2019). Lack of vimentin results in failure of C3bot to foster axonal outgrowth in hippocampal/neocortical neurons as shown in vim −/− cultures (Adolf et al., 2016). ...
... GST was removed by thrombin cleavage. Vimentin expression and purification were performed as described before (Rohrbeck et al., 2014). Plasmids of full-length mouse vimentin provided by Prof. Dr. Yi-Ling Li, Institute of Biomedical Sciences, Genomics Research Center, Academia Sinica, Taipei, Taiwan were used. ...
... A monoclonal rat anti-ß1-integrin IgG (#MAB 2405, IF dilution 1:500) was from R&D Systems (Minneapolis, MN, USA). An affinity-purified polyclonal rabbit IgG against full-length C3bot (IF dilution 1:1,000, WB dilution 1:2,000) was developed in our lab (Rohrbeck et al., 2014). A monoclonal mouse anti-glyceraldehyde-3phosphate dehydrogenase IgG (GAPDH, MAB374, WB dilution 1:4,000, Dazard et al., 2003) was from Merck Millipore. ...
The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of β1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects. © Copyright © 2020 Adolf, Turko, Rohrbeck, Just, Vida, Ahnert-Hilger and Höltje.
... This includes experimental lesion models both in vitro and in vivo (Dubreuil et al., 2003;Höltje et al., 2009;Boato et al., 2010;Loske et al., 2012) as well as clinical trials in patients with spinal cord injuries (McKerracher and Anderson, 2013;Watzlawick et al., 2014;Fehlings et al., 2018). For a long time unknown, recent work from our group has identified the intermediate filament protein vimentin as well as β1-integrin as cellular receptors for C3bot (Rohrbeck et al., 2014(Rohrbeck et al., , 2017Adolf et al., 2019). As mentioned, growth-promoting effects are also caused by enzyme-deficient C3bot-derived peptides like the 29mer C3bot 154−182 or 26mer C3bot 156−181 , both including the catalytic amino acid glutamate at position 174 of full length C3bot, but in this case, the exact cellular enzyme-independent mechanisms remain to be unraveled. ...
... Morphology of hippocampal neurons was visualized by a polyclonal antiserum against microtubule associated protein 2 (MAP2, #AB5622) and neurofilament protein of 200 kDa (#AB5256) from Chemicon International (Hofheim, Germany). An affinity purified polyclonal rabbit IgG against full length C3bot developed by our group was applied (Rohrbeck et al., 2014). To detect the phosphorylation levels of EAAT3 a polyclonal antibody directed against phosphotyrosine was purchased from Santa Cruz Biotechnology (#sc-7020). ...
In primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot156–181 peptide resulted in the upregulation of glutamate uptake by up to 22% compared to untreated cells. A similar enhancement of glutamate transport was also achieved by the classical phorbol-ester-mediated activation of protein kinase C subtypes. Yet comparable, effects elicited by C3 preparations seemed not to rely on PKCα, γ, ε, or ζ activation. Blocking of tyrosine phosphorylation by tyrosine kinase inhibitors prevented the observed effect mediated by C3bot and C3bot 26mer. By using biochemical and molecular biological assays we could rule out that the observed C3bot and C3bot 26mer-mediated effects solely resulted from enhanced transporter expression or translocation to the neuronal surface but was rather mediated by transporter phosphorylation at tyrosine residues that was found to be significantly enhanced following incubation with either full length protein or the 26mer C3 peptide.
... Binding of NnPLA 2 -I to vimentin resulted in its internalization into partially differentiated myoblasts. The involvement of vimentin in cellular uptake mechanisms has already been shown for C3, a Clostridium botulinum toxin [84,85], and several viruses, such as human immunodeficiency virus type 1, vaccinia virus and the severe acute respiratory syndrome coronavirus [86]. In all of these cases, vimentin acts as a component of the cellular attachment mechanism, either as a receptor or a co-receptor. ...
... In a similar way, vimentin might mediate internalization of sPLA2s. This is further supported by the similar location of the binding sites on vimentin for dengue virus DENV-2 envelope protein domain III, Clostridium botulinum C3 exoenzyme and NnPLA 2 -I, all of which have been proposed to bind to the rod domain of vimentin [26,84,87]. ...
Secreted phospholipases A2 (sPLA2s) participate in a very broad spectrum of biological processes through their enzymatic activity and as ligands for membrane and soluble receptors. The physiological roles of sPLA2s as enzymes have been very well described, while their functions as ligands are still poorly known. Since the last overview of sPLA2-binding proteins (sPLA2-BPs) 10 years ago, several important discoveries have occurred in this area. New and more sensitive analytical tools have enabled the discovery of additional sPLA2-BPs, which are presented and critically discussed here. The structural diversity of sPLA2-BPs reveals sPLA2s as very promiscuous proteins, and we offer some structural explanations for this nature that makes these proteins evolutionarily highly advantageous. Three areas of physiological engagement of sPLA2-BPs have appeared most clearly: cellular transport and signalling, and regulation of the enzymatic activity of sPLA2s. Due to the multifunctionality of sPLA2s, they appear to be exceptional pharmacological targets. We reveal the potential to exploit interactions of sPLA2s with other proteins in medical terms, for the development of original diagnostic and therapeutic procedures. We conclude this survey by suggesting the priority questions that need to be answered.
... Alternatively, the toxin can be injected manually. However, binding to the type III intermediate filament protein vimentin has been suggested to be sufficient for cell entry into damaged neurons [41]. ...
Pathogenic bacteria produce diverse protein toxins to disturb the host’s defenses. This includes the opening of epithelial barriers to establish bacterial growth in deeper tissues of the host and to modulate immune cell functions. To achieve this, many toxins share the ability to enter mammalian cells, where they catalyze the modification of cellular proteins. The enzymatic activity is diverse and ranges from ribosyl- or glycosyl-transferase activity, the deamidation of proteins, and adenylate-cyclase activity to proteolytic cleavage. Protein toxins are highly active enzymes often with tight specificity for an intracellular protein or a protein family coupled with the intrinsic capability of entering mammalian cells. A broad understanding of their molecular mechanisms established bacterial toxins as powerful tools for cell biology. Both the enzymatic part and the pore-forming/protein transport capacity are currently used as tools engineered to study signaling pathways or to transport cargo like labeled compounds, nucleic acids, peptides, or proteins directly into the cytosol. Using several representative examples, this review is intended to provide a short overview of the state of the art in the use of bacterial toxins or parts thereof as tools.
... Clostridia, Bacilli, and Staphylococci produce the toxin exoenzyme C3 (Just et al., 1992;Wilde et al., 2001;Wilde et al., 2003). Devoid of binding and translocation domains, its apparent mode of entry is via vimentin-dependent endocytosis (Rohrbeck et al., 2014). It is an ADP-ribosylating transferase which catalyses the hydrolysis of nicotinamide adenine dinucleotide (NAD) ...
The heterotrimeric G-proteins are critically important intracellular signalling
molecules that regulate fundamental processes in cellular homeostasis. A
unique bacterial toxin, Pasteurella multocida toxin (PMT) modifies 3 of the 4
families of G-proteins (Gq, Gi, and G12) by deamidation which leads to a
plethora of downstream signalling. PMT induces drastic morphological
changes such as loss of adherence to the matrix, foci and stress fibre
formation in Swiss 3T3 and Vero cells. PMT is mitogenic for many cell types
but not all, and the work reported in this thesis aims to compare two cell
lines that respond differently to PMT.
Swiss 3T3 and Vero cells were treated with different concentrations of PMT
to determine the effects on cell proliferation, cytoskeletal reorganisation and
cell death. PMT induced prominent cytoskeletal changes, mitogenic and
anti-cell death responses in Swiss 3T3 cells while delayed cytoskeletal
changes with no evidence of mitogenicity and cell death were observed in
Vero cells. PMT modified G-proteins at different times in Swiss 3T3 and
Vero cells. The PMT-induced anti-cell death response in Swiss 3T3 cells
was dose-dependent while the delayed cytoskeletal response in Vero cells
was linked to the late PMT-mediated G12 activation. The amino acid
sequence of Gα12 differed in the two cell types – the G12 subunit in Vero
cells is missing N-terminal cysteine residue, which may have contributed to
the differences. Gq/11 signalling is active and sustained in Swiss 3T3 cells,
but not in Vero cells. G may have inhibited adenylyl cyclase activity so it is
unknown whether Gi signalling is active and sustained over the 3-day period
as the forskolin-stimulated cAMP level decreased in both serum-starved
untreated and PMT-treated cell lines. In summary, I have identified changes
in both the primary effect of PMT on the two cell types and also on
downstream signalling that is likely to reflect the differential cell response.
... To date, very little is known about the cellular uptake of C3 toxins. Some research has suggested that C3 toxins are internalized into endosomes and traverse into the cytoplasm during the acidification process; others propose that C3 toxins bind membrane-bound vimentin using an RGD motif to gain access to the cell (47,49). Currently, the only widely accepted theory is that these toxins are selectively internalized and inevitably locate to cytoplasmic Rho-GTPases. ...
C3larvinA was recently described as a mono-ADP-ribosyltransferase toxin from the ERIC III genotype of the agricultural pathogen, Paenibacillus larvae. It was shown to be the full-length, functional version of the previously described C3larvintrunc toxin, due to a 33-residue extension of the N-terminus of the protein. In this study, a series of deletions and substitutions were made to the N-terminus of C3larvinA to assess the contribution of the α1-helix to toxin structure and function. Catalytic characterization of these variants identified Asp23 and Ala31 residues as supportive to enzymatic function. A third residue, Lys36, was also found to contribute to the catalytic activity of the enzyme. Analysis of the C3larvinA homology model revealed that these three residues were participating in a series of interactions to properly orient both the Q-X-E and S-T-S motifs. Ala31 and Lys36 were found to associate with a structural network of residues previously identified in silico, whereas Asp23 forms novel interactions not previously described. Lastly, the membrane translocation activity into host target cells of each variant was assessed, highlighting a possible relationship between protein dipole and target cell entry.
... C3 toxins are internalized most likely into the endosomes of these cells and released into their cytosol, which is probably triggered by the acidification of maturating endosomes [11]. Furthermore, it has recently been shown that intermediate filament protein vimentin may also play a role in the uptake of C3 toxins in target cells [13,14]. However, the exact mechanism is not clarified yet and it is not known whether vimentin acts as a receptor to trigger endosomal uptake. ...
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.
Unlike other cholera‐like toxins that contain separate binding/translocation and catalytic subunits, C3‐like mono‐ADP‐ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently labeled C3larvinA. Following binding to the macrophage membrane in a discontinuous punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA‐treated cells underwent massive swelling over the course of 1–4 h. Lysosomal swelling preceded the extensive rearrangement of the cellular F‐actin caused by ADP‐ribosylation of cytosolic Rho‐GTPases. This suggested that lysosome swelling might be required for the escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F‐actin rearrangement. Toxin‐induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano‐sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodeling activities of C3larvinA with that of two related toxins, C3larvintrunc and Plx2A, highlighted the importance of the N‐terminal α1‐helix for lysosomal swelling and successful intoxication.
Unlike other cholera-like toxins that contain separate binding/translocation and catalytic subunits, C3-like mono-ADP-ribosyltransferases consist of a single subunit that serves both functions. The manner whereby C3 toxins reach the host cell cytoplasm is poorly understood and was addressed in this study by monitoring the fate of fluorescently-labelled C3larvinA. Following binding to the macrophage membrane in a discontinuous, punctate pattern, the toxin was internalized, traversing the endocytic pathway to reach lysosomes. Strikingly, the lysosomes of C3larvinA-treated cells underwent massive swelling over the course of 1-4 hrs. Lysosomal swelling preceded the extensive rearrangement of the cellular F-actin caused by ADP-ribosylation of cytosolic Rho-GTPases. This suggested that lysosome swelling might be required for escape of the toxin into the cytoplasm where the GTPases reside. Accordingly, preventing swelling by osmotic manipulation or by arresting macropinocytosis precluded the F-actin rearrangement. Toxin-induced swelling was associated with leakage of sulforhodamine B and dextran from the lysosomes, implying membrane rupture or activation of mechano-sensitive pores, enabling the toxin itself to reach the cytosol. Finally, comparison of the cellular traffic and actin remodelling activities of C3larvinA with that of two related toxins, C3larvin trunc and Plx2A, highlighted the importance of the N-terminal α 1 -helix for lysosomal swelling and successful infection.
Extracellular matrix (ECM) stiffness is one of many mechanical forces acting on mammalian adherent cells that influence cellular function. We have addressed the open question of how ECM stiffness might alter the susceptibility of host cells to infection by bacterial pathogens. We manufactured hydrogels of varying physiologically-relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm) and found that adhesion of Lm onto host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1, and demonstrated that bacterial infection of these host cells is decreased when surface vimentin is perturbed. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of host cells to bacterial infection.
We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and-37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ribosylation had no major effects on GDP release from RhoA, Rac1, and Cdc42, the rate of GTPγS release from Rho proteins was increased 3-6-fold by glucosylation. ADP ribosylation decreased the rate of GTPγS release by about 50%. Glucosylation reduced the intrinsic activities of the GTPases by 3-7-fold and completely blocked GTPase stimulation by Rho-GAP. In contrast, ADP ribosylation slightly increased GTPase activity (∼2-fold) and had no major effect on GAP stimulation of GTPase. Whereas ADP ribosylation did not affect the interaction of RhoA with the binding domain of protein kinase N, glucosylation inhibited this interaction. Glucosylation of Rac1 markedly diminished its ability to support the activation of the superoxide-generating NADPH oxidase of phagocytes. Glucosylated Rac1 did not interfere with NADPH oxidase activation by unmodified Rac1, even when present in marked molar excess, indicating that it was incapable of competing for a common effector. The data indicate that the functional inactivation of small GTPases by glucosylation is mainly caused by inhibition of GTPase-effector protein interaction.
A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin into and through the Golgi apparatus.
Peripheral nerve injury triggers the activation of RhoA in spinal motor and peripheral sensory neurons. RhoA activates a number of effector proteins including the Rho-associated kinase, ROCK, which targets the cytoskeleton and leads to inhibition of neurite outgrowth. Blockade of the Rho/ROCK pathway by pharmacological means improves axon regeneration after experimental injury. C3bot transferase, an exoenzyme produced by Clostridium botulinum, inactivates RhoA by ADP-ribosylation. Up to now it was not investigated thoroughly whether C3bot exerts positive effects on peripheral axon regeneration as well. In the present study, recombinant membrane permeable C3bot produced a small, but significant, axon outgrowth effect on peripheral sensory neurons dissociated from adult dorsal root ganglia of the rat. Neuronal overexpression of C3, however, did not enhance axonal growth. Moreover, transfection of plasmids encoding dominant negative RhoA or RhoA specific shRNAs failed to increase axonal growth. Furthermore, we show that the C3bot mutant, C3E174Q, which lacks RhoA inhibitory activity, still stimulates axonal growth. When analyzing possible signaling mechanisms we found that ERK (extracellular signal-regulated kinase) and Akt are activated by C3bot and ERK is induced by the C3E174Q mutant. Upregulation of kinase activities by C3bot occurs significantly faster than inactivation of RhoA indicating a RhoA-independent pathway of action by C3bot. The induction of ERK signaling by C3bot was detected in embryonic hippocampal neurons, too. Taken together, although RhoA plays a central role for inhibition of axon outgrowth by myelin-derived inhibitors, it does not interfere with axonal growth of sensory neurons on a permissive substrate in vitro. C3bot blocks neuronal RhoA activity, but its positive effects on axon elongation and branching appear to be mediated by Rho independent mechanisms involving activation of axon growth promoting ERK and Akt kinases.
Background and purpose:
In the spinal cord injury (SCI) axon regeneration is inhibited by the glial scar, which contains reactive astrocytes that secrete inhibitory chondroitin sulphate proteoglycan (CSPG). We previously reported that a novel compound, denosomin, promotes axonal growth under degenerative conditions in cultured cortical neurons. In this study, we investigated the effects of denosomin on functional recovery in SCI mice and elucidated the mechanism though which denosomin induces axonal growth in the injured spinal cord.
Experimental approach:
Denosomin was administered p.o. for 7 or 14 days to contusion mice. Behavioural evaluations and immunohistochemistry were done. Primary cultured cortical neurons and astrocytes were treated with denosomin to investigate the mechanism of axonal growth facilitation.
Key results:
Denosomin improved hind limb motor dysfunction and axonal growth, especially in the 5-HT-positive tracts across the scar and increased the density of astrocytes. Denosomin increased astrocyte proliferation, inhibited astrocytic death and increased the expression and secretion of vimentin in cultured astrocytes. Furthermore, vimentin increased axonal outgrowth in cultured neurons, even in the presence of inhibitory CSPG. Denosomin increased the number of vimentin-expressing astrocytes inside glial scars of SCI mice, and 5-HT-positive axonal growth occurred in a vimentin-associated manner.
Conclusion and implications:
Denosomin increased the ratio of astrocytes that secrete vimentin as an axonal growth facilitator, which, we propose enhances axonal growth beyond the glial scar and promotes functional recovery in SCI mice. This study is the first to demonstrate this novel role of vimentin in SCI and drug-mediated modification of the inhibitory property of reactive astrocytes.
Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.
C3-like exoenzymes are produced by various microorganism including Clostridium botulinum (C3bot), Bacillus cereus and Staphylococcus aureus. C3bot is the prototype of C3-like exoenzymes that specifically ADP-ribosylates and thereby inactivates Rho(A/B/C). C3-like exoenzymes are not yet regarded as virulence factors, as the lack of cell entry domains results in a poor accessibility of the C3-like exoenzymes to cells. In this study, the sensitivity of various cell lines to C3bot has been reinvestigated. Primary monocytes as well as cultured macrophage-like cells including J774A.1 cells and RAW macrophages exhibit a tenfold higher sensitivity to C3bot than fibroblasts and epithelial cells. RhoA ADP-ribosylation by C3bot resulted in the formation of pronounced bipolar protrusions based on defective tail retraction. The formation of bipolar protrusion resulted in inhibited macrophage migration. These findings suggested that macrophages appear to be target cells of C3bot. Migration of macrophage is a prerequiste for their recruitment to the site of pathogen invasion or tissue damage. Inhibition of macrophage migration likely preserves the survival of C3-producing microorganisms. The observations of this study reinforce the paradigm of a role of C3-like exoenzymes as virulence factors.
This study describes the development and use of a specific method for disassembling intermediate filament (IF) networks in living cells. It takes advantage of the disruptive effects of mimetic peptides derived from the amino acid sequence of the helix initiation 1A domain of IF protein chains. The results demonstrate that at 1:1 molar ratios, these peptides disassemble vimentin IF into small oligomeric complexes and monomers within 30 min at room temperature in vitro. Upon microinjection into cultured fibroblasts, these same peptides induce the rapid disassembly of IF networks. The disassembly process is accompanied by a dramatic alteration in cell shape and the destabilization of microtubule and actin-stress fiber networks. These changes in cell shape and IF assembly states are reversible. The results are discussed with respect to the roles of IF in cell shape and the maintenance of the integrity and mechanical properties of the cytoplasm, as well as the stability of the other major cytoskeletal systems.
Peripheral nerve injury triggers the activation of the small GTPase RhoA in spinal motor and peripheral sensory neurons. C3 transferase, an exoenzyme produced by Clostridium botulinum that inactivates RhoA by ADP-ribosylation, has been successfully applied in central nervous system (CNS) lesion models to facilitate regeneration functionally and morphologically. Until now it has not been demonstrated if C3bot exerts positive effects on peripheral axon regeneration as well. In organotypic spinal cord preparations, C3bot reduced axonal growth of motoneurons, while no effect on sensory axon outgrowth from dorsal root ganglia (DRG) explants was observed. Enzymatically inactive C3E174Q was ineffective in both culture models. Spinal cord slices exhibited a significant increase in microglia/macrophages after treatment with C3bot suggesting an inflammatory component in the inhibition of axon growth. C3bot or C3E174Q were then applied into conduits implanted after transection of the sciatic nerve in rats. Functional evaluation by electrophysiology, nociception, and walking track tests did not show any significant difference between groups with active or mutant C3E174Q . Transmission electron microscopy of the regenerated nerves revealed no significant differences in the number of myelinated and unmyelinated axons 6 weeks after surgery. Compared to the CNS, the functional significance of RhoA may be limited during nerve regeneration in a growth-promoting environment.
Salmonella has evolved an intimate functional interface with its host. Central to this interface is a battery of bacterial proteins delivered into host cells via a specialized organelle termed the type III secretion system. A subset of these bacterial proteins stimulates cellular responses by activating the Rho family GTPases Cdc42 and Rac. Stimulation of these responses leads to actin cytoskeleton reorganization and the activation of cellular transcription factors that result in bacterial uptake and proinflammatory cytokine production. Remarkably, the cellular responses stimulated by Salmonella are quickly reversed by another bacterial protein, SptP, which exerts its function as a GTPase-activating protein (GAP) for Cdc42 and Rac. In addition to its GAP activity located within its amino-terminus, the carboxy-terminal domain of SptP possesses potent tyrosine phosphatase activity. We show here that the tyrosine phosphatase activity of SptP is involved in reversing the MAP kinase activation that results from Salmonella infection. We also demonstrate an important role for tyrosine kinases, including ACK, in the cellular responses induced by Salmonella. We also found that a potential target for the tyrosine phosphatase activity of SptP is the intermediate filament protein vimentin, which is recruited to the membrane ruffles stimulated by Salmonella.
Low-molecular-weight GTP-binding proteins of the Rho family control the organization of the actin cytoskeleton in eukaryotic cells. Dramatic reorganization of the actin cytoskeleton is caused by the C3 exoenzyme derived from Clostridium botulinum (C3), based on ADP-ribosylation of RhoA/B/C. In addition, wild-type as well as ADP-ribosyltransferase-deficient C3-E174Q induce axonal outgrowth of primary murine hippocampal neurons and prevent growth cone collapse, indicating a non-enzymatic mode of action. In this study, we compared the effects of C3-E174Q and wild-type C3 in the murine hippocampal cell line HT22. Treatment of HT22 cells with C3 resulted in Rho ADP-ribosylation and cell rounding. The ADP-ribosyltransferase-deficient mutant C3-E174Q did not induce either Rho ADP-ribosylation or morphological changes. C3 as well as C3-E174Q treatment resulted in growth arrest, reduced expression of cyclin D levels, and increased expression of RhoB, a negative regulator of cell-cycle progression. Serum starvation induced apoptosis in HT22 cells, as determined on the basis of increased expression of caspase-9 and Bax. C3 but not C3-E174Q protected serum-starved HT22 cells from apoptosis. This is the first study separating ADP-ribosyltransferase-dependent from ADP-ribosyltransferase-independent effects of C3. While morphological changes and anti-apoptotic activity strictly depend on ADP-ribosyltransferase activity, the anti-proliferative effects are independent of ADP-ribosyltransferase activity.
Structured digital abstract