Clostridium botulinum C3bot mediated effects on cytokine-induced psoriasis-like phenotype in full-thickness skin model.

Clostridium botulinum C3 exoenzyme (C3bot) exclusively inhibits RhoA, B and C by ADP-ribosylation and is therefore used as a cell-permeable tool for investigating the cellular role of these Rho-GTPases. Rho-GTPases represent a molecular switch integrating different receptor signalling to downstream cascades including transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton and cell proliferation. C3bot-induced inhibition of RhoA leads to reorganization of the actin cytoskeleton, morphological changes, and inhibition of cell proliferation as well as modulation of inflammatory response. In this study, we characterized the C3bot-mediated effects on a full-thickness skin model exhibiting a psoriasis-like phenotype through the addition of cytokines. Indeed, after the addition of cytokines, a decrease in epidermal thickness, parakeratosis, and induction of IL-6 was detected. In the next step, it was studied whether C3bot caused a reduction in the cytokine-induced psoriasis-like phenotypes. Basal addition of C3bot after cytokine induction of the full-thickness skin models caused less epidermal thinning and reduced IL-6 abundance. Simultaneous basal incubation with cytokines and C3bot, IL-6 abundance was inhibited, but epidermal thickness was only moderately affected. When C3bot was added apically to the skin model, IL-6 abundance was reduced, but no further effects on the psoriasis-like phenotype of the epidermis were observed. In summary, C3bot inhibits the cytokine-induced expression of IL-6 and thus may have an impact on the pro-inflammatory immune response in the psoriasis-like phenotype.

MS-based quantification of RhoA/B and RhoC ADP-ribosylation.

Mono ADP-ribosylation is a common characteristic of bacterial toxins resulting to aberrant activation or inactivation of target proteins. The C3 exoenzyme of Clostridium botulinum (C3bot) ADP-ribosylates the small GTPases RhoA, RhoB and RhoC, leading to inactivation of these important regulators and impaired down-stream signaling. Quantification of ADP-ribosylation using gel migration assays, antibodies, and radioactivity-based methods are limited. Therefore a novel LC-MS-based method to specifically determine and quantify ADP-ribosylation of Rho GTPases was established. A heavy labeled protein standard that contained ADP-ribosylation specific peptides in similar amounts in ADP ribosylated and non ADP ribosylated form was used for relative quantification in vivo. In a proof of principle experiment HT22 cells were treated with C3bot and the kinetics of RhoA/B and RhoC ADP-ribosylation were determined in vivo.

Glucosyltransferase-dependent and -independent effects of TcdB on the proteome of HEp-2 cells.

Toxin B (TcdB) of the nosocomial pathogen C. difficile has been reported to exhibit a glucosyltransferase-dependent and -independent effect on treated HEp-2 cells at toxin concentration above 0.3 nM. In order to investigate and further characterize both effects epithelial cells were treated with wild type TcdB and glucosyltransferase-deficient TcdBNXN and their proteomes were analyzed by LC-MS. Triplex SILAC labeling was used for quantification. Identification of 5212 and quantification of 4712 protein groups was achieved. Out of these 257 were affected by TcdB treatment, 92 by TcdBNXN treatment and 49 by both. TcdB mainly led to changes in proteins that are related to "GTPase mediated signaling" and the "cytoskeleton" while "chromatin" and "cell cycle" related proteins were altered by both, TcdB and TcdBNXN . The obtained dataset of HEp-2 cell proteome helps us to better understand glucosyltransferase-dependent and -independent mechanisms of TcdB and TcdBNXN , particularly those involved in pyknotic cell death. All proteomics data have been deposited in the ProteomeXchange with the dataset identifier PXD006658 (https://proteomecentral.proteomexchange.org/dataset/PXD006658).

Toxin A of the nosocomial pathogen Clostridium difficile induces primary effects in the proteome of HEp-2 cells.

PURPOSE: This study was carried out to investigate the impact of high concentrations of Clostridium difficile toxin A (TcdA) on the proteome of human cells. It should also be examined whether a catalytically deficient mutant (TcdANXN ) has an effect on target cells. EXPERIMENTAL DESIGN: Proteome changes were investigated after treatment of HEp-2 cells with 20 nM TcdA for 8 h using a triplex SILAC labeling method and shotgun proteomics. Proteins from differently labeled and treated cells were combined for analysis using an HPLC coupled to an Orbitrap mass spectrometer. RESULTS: Nearly 4000 proteins were identified in each replicate and 3500 could be quantified by SILAC triplicate analysis. 51 proteins exhibited an altered abundance with 29 up-regulated and 22 down-regulated proteins. In contrast, TcdANXN had no provable impact on the protein profile of HEp-2 cells. Data analysis of regulated proteins revealed that mainly plasma membrane, cell death, cell proliferation and actin cytoskeleton proteins were affected by TcdA treatment. CONCLUSIONS AND CLINICAL RELEVANCE: This proteome analysis showed novel insights of TcdA impact onepithelial cells. Comparison with long-term treatment studies reveals distinctions in affected cellular processes that will improve the understanding of TcdA functions and might help to find new tools for diagnosis and treatment of CDI.

The intermediate filament protein vimentin is essential for axonotrophic effects of Clostridium botulinum C3 exoenzyme.

The type III intermediate filament protein vimentin was recently identified to mediate binding and uptake of Clostridium botulinum C3 exoenzyme (C3bot) in two cell lines. Here, we used primary neuronal cultures from vimentin knockout (Vim-/- ) mice to study the impact of vimentin on axonal growth and internalization of C3bot. In contrast to wild type, vimentin knockout neurons were insensitive to C3bot. Application of extracellular vimentin to Vim-/- neurons completely restored the growth-promoting effects of C3bot. In line with this uptake of C3bot into Vim-/- neurons was strongly decreased resulting in reduced ADP-ribosylation of RhoA and B as detected by an antibody recognizing selectively ADP-ribosylated RhoA/B. Again, uptake of C3bot into Vim-/- neurons was rescued by addition of extracellular vimentin. In addition, in purified embryonic stem cell-derived motor neurons that are devoid of glial cells C3bot elicited axonotrophic effects confining neuronal vimentin as a binding partner. Primary neuronal cultures from vimentin knockout (KO) mice were used to study the impact of vimentin on axonal growth and internalization of C3bot. In contrast to wild type, vimentin knockout neurons were insensitive to the axonotrophic effects of C3bot. Application of extracellular vimentin (recombinant vimentin) to vimentin KO neurons completely restored the growth-promoting effects of C3bot. In line with this uptake of C3bot into vimentin KO neurons was strongly decreased resulting in reduced ADP-ribosylation of RhoA and B as detected by an antibody recognizing selectively ADP-ribosylated RhoA/B.

C3 exoenzyme impairs cell proliferation and apoptosis by altering the activity of transcription factors.

C3 exoenzyme from C. botulinum is an ADP-ribosyltransferase that inactivates selectively RhoA, B, and C by coupling an ADP-ribose moiety. Rho-GTPases are involved in various cellular processes, such as regulation of actin cytoskeleton, cell proliferation, and apoptosis. Previous studies of our group with the murine hippocampal cell line HT22 revealed a C3-mediated inhibition of cell proliferation after 48 h and a prevention of serum-starved cells from apoptosis. For both effects, alterations of various signaling pathways are already known, including also changes on the transcriptional level. Investigations on the transcriptional activity in HT22 cells treated with C3 for 48 h identified five out of 48 transcription factors namely Sp1, ATF2, E2F-1, CBF, and Stat6 with a significantly regulated activity. For validation of identified transcription factors, studies on the protein level of certain target genes were performed. Western blot analyses exhibited an enhanced abundance of Sp1 target genes p21 and COX-2 as well as an increase in phosphorylation of c-Jun. In contrast, the level of p53 and apoptosis-inducing GADD153, a target gene of ATF2, was decreased. Our results reveal that C3 regulates the transcriptional activity of Sp1 and ATF2 resulting downstream in an altered protein abundance of various target genes. As the affected proteins are involved in the regulation of cell proliferation and apoptosis, thus the C3-mediated anti-proliferative and anti-apoptotic effects are consequences of the Rho-dependent alterations of the activity of certain transcriptional factors.

Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.

MALDI imaging mass spectrometry to investigate endogenous peptides in an animal model of Usher's disease.

Imaging MS (MSI) has emerged as a valuable tool to study the spatial distribution of biomolecules in the brain. Herein, MALDI-MSI was used to determine the distribution of endogenous peptides in a rat model of Usher's disease. This rare disease is considered as a leading cause of deaf-blindness in humans worldwide. Cryosections of brain tissue were analyzed by MALDI-MSI to differentiate between healthy and diseased rats. MSI results were highly reproducible. Tissue-specific peptides were identified by MS/MS using LC-Orbitrap and MALDI-TOF/TOF analyses. These peptides were proposed for histological classification due to their particular spatial distribution in the brain, for example, substantia nigra, corpus callosum, and hippocampus. Several endogenous peptides showed significantly increased ion densities, particularly in the colliculi superiores and in the substantia nigra of diseased rats, including peptides derived from Fsd1, dystrobrevin-ß, and ProSAAS. Furthermore, several proteolytic degradation products of the myelin basic protein were identified, of which one peptide is most likely mediated by calpain-2. Our findings contribute to the characterization of this animal model and include possible peptide markers of disease.

Expression and cytoprotective activity of the small GTPase RhoB induced by the Escherichia coli cytotoxic necrotizing factor 1.

RhoB is the only member of the Rho subfamily of small GTPases, which is classified as an immediate early gene product. RhoB is up-regulated in response to growth factors as well as cytotoxic and genotoxic agents. Clostridial glucosylating toxins have been reported to evoke pronounced RhoB expression, based on the inactivation of Rho/Ras proteins. In this study, we report on a long lasting expression of RhoB in cultured cells upon activation of Rho proteins by the cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. The observations of this study highlight a new pathway involving Rac1, which positively regulates the activity of the rhoB promoter and RhoB expression. Conversely, the isomeric cytotoxic necrotizing factor from Yersinia pseudotuberculosis (CNFy) drives GTP-loading of basal RhoB but fails to cause activation of the rhoB promoter and thus its expression. CNF1 inhibits cytokinesis and induces the formation of bi-nucleated (tetraploid) cells. Upon long term treatment with CNF1, RhoB(-/-) mouse embryonic fibroblasts (MEFs) exhibit DNA fragmentation, phosphatidylserine exposure, and loss of membrane integrity, while RhoB(+/-) MEFs persist as bi-nucleated (tetraploid) cells without any signs of cell death. In conclusion, the cytoprotective RhoB response is not only evoked by bacterial protein toxins inactivating Rho/Ras proteins but also by the Rac1-activating toxin CNF1.

Inhibition of macrophage migration by C. botulinum exoenzyme C3.

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.

Minimal essential length of Clostridium botulinum C3 peptides to enhance neuronal regenerative growth and connectivity in a non-enzymatic mode.

C3 ADP-ribosyltransferase is a valuable tool to study Rho-dependent cellular processes. In the current study we investigated the impact of enzyme-deficient peptides derived from Clostridium botulinum C3 transferase in the context of neuronal process elongation and branching, synaptic connectivity, and putative beneficial effects on functional outcome following traumatic injury to the CNS. By screening a range of peptidic fragments, we identified three short peptides from C3bot that promoted axon and dendrite outgrowth in cultivated hippocampal neurons. Furthermore, one of these fragments, a 26-amino acid peptide covering the residues 156-181 enhanced synaptic connectivity in primary hippocampal culture. This peptide was also effective to foster axon outgrowth and re-innervation in organotypical brain slice culture. To evaluate the potential of the 26mer to foster repair mechanisms after CNS injury we applied this peptide to mice subjected to spinal cord injury by either compression impact or hemisection. A single local administration at the site of the lesion improved locomotor recovery. In addition, histological analysis revealed an increased serotonergic input to lumbar motoneurons in treated compared with control mice. Pull-down assays showed that lesion-induced up-regulation of RhoA activity within the spinal cord was largely blocked by C3bot peptides despite the lack of enzymatic activity.

C3 peptide promotes axonal regeneration and functional motor recovery after peripheral nerve injury.

Peripheral nerve injuries are frequently seen in trauma patients and due to delayed nerve repair, lifelong disabilities often follow this type of injury. Innovative therapies are needed to facilitate and expedite peripheral nerve regeneration. The purpose of this study was to determine the effects of a 1-time topical application of a 26-amino-acid fragment (C3(156-181)), derived from the Clostridium botulinum C3-exoenzyme, on peripheral nerve regeneration in 2 models of nerve injury and repair in adult rats. After sciatic nerve crush, different dosages of C3(156-181) dissolved in buffer or reference solutions (nerve growth factor or C3(bot)-wild-type protein) or vehicle-only were injected through an epineurial opening into the lesion sites. After 10-mm nerve autotransplantation, either 8.0 nmol/kg C3(156-181) or vehicle were injected into the proximal and distal suture sites. For a period of 3 to 10 postoperative weeks, C3(156-181)-treated animals showed a faster motor recovery than control animals. After crush injury, axonal outgrowth and elongation were activated and consequently resulted in faster motor recovery. The nerve autotransplantation model further elucidated that C3(156-181) treatment accounts for better axonal elongation into motor targets and reduced axonal sprouting, which are followed by enhanced axonal maturation and better axonal functionality. The effects of C3(156-181) are likely caused by a nonenzymatic down-regulation of active RhoA. Our results indicate the potential of C3(156-181) as a therapeutic agent for the topical treatment of peripheral nerve repair sites.

Down-regulation of interleukin-16 in human mast cells HMC-1 by Clostridium difficile toxins A and B.

Toxin A (TcdA) and toxin B (TcdB) are the major virulence factors of Clostridium difficile and are the causative agents for clinical symptoms, such as secretory diarrhoea and pseudomembranous colitis. Mast cells are essentially involved in the toxin-induced colonic inflammatory processes. To study the direct effects of these toxins on the expression of inflammatory genes, a DNA microarray containing evaluated probes of 90 selected inflammatory genes was applied to the immature mast cell line HMC-1. TcdA and TcdB induced up-regulation of only a limited number of genes within the early phase of cell treatment. Interleukin-8 (IL-8), transcription factor c-jun and heme oxygenase-1 messenger RNA (mRNA) increased more than 2-fold. In contrast, IL-16, known as a CD4(+) T-cell chemoattractant factor and the chemokine receptor cKit were down-regulated. Stimulation of HMC-1 cells with IL-8 had no effect on IL-16 mRNA level, indicating that both cytokines were independently affected by the toxins. Regulation of both cytokines, however, depended on glucosylation of Rho GTPases as tested by application of enzyme-deficient TcdA or TcdB. Down-regulation of total and secreted IL-16 protein was checked by enzyme-linked immunosorbent assay. The data implicate that TcdA and TcdB affect lymphocyte migration by modulating release of the chemoattractant factor IL-16 from mast cells. In addition, this is the first report showing that Rho GTPases are involved in the regulation of IL-16 expression.

Functional implications of lethal toxin-catalysed glucosylation of (H/K/N)Ras and Rac1 in Clostridium sordellii-associated disease.

Clostridium sordellii-based diseases in humans and livestock rely on the activity of the major virulence factors, the single-chain protein toxins TcsL and TcsH, both belonging to the large clostridial glucosylating toxins. TcsL exclusively glucosylates Rho and Ras low molecular weight GTP-binding proteins. TcsL-induced loss of barrier function in epithelial (diarrhoea) and endothelial cells (extravasation of blood fluid) is based on Rac glucosylation whereas induction of apoptosis results from glucosylation of Ras. Intracellular glucosylation of Rac and Ras can be tracked by immunoblot applying the glucosylation-sensitive antibodies Rac1(Mab 102) and Ras(Mab 27H5). Induction of apoptosis especially of phagocytotic cells is crucial for the severity of C. sordellii-associated disease. The inhibition of TcsL-induced apoptosis by tauroursodeoxycholic acid (TUDCA) may be a promising therapeutic option.

Autoproteolytic cleavage mediates cytotoxicity of Clostridium difficile toxin A.

Toxin A and toxin B from Clostridium difficile are the causative agents of the antibiotic-associated pseudomembranous colitis. They are of an A/B structure type and possess inositol hexakisphosphate-inducible autoproteolytic activity to release their glucosyltransferase domain to the cytoplasm of target cells. In this study, we investigated the effect of extracellular and intracellular autoproteolytic cleavage on the function of TcdA. Extracellular cleavage led to functional inactivation albeit TcdA was less susceptible to inositol hexakisphosphate-induced autoproteolysis than TcdB. A non-cleavable TcdA mutant (TcdA A541 G542 A543) was generated to investigate whether autoproteolysis is a prerequisite for intracellular function of TcdA. Although the EC(50) regarding cell rounding was about 75-fold reduced in short-term assay, non-cleavable TcdA was able to induce complete cell rounding and apoptosis after 36 h comparable to wildtype TcdA when continuously present. Studies with limited uptake of toxins revealed progressive Rac1 glucosylation and complete cell rounding for TcdA, whereas the effect induced by non-cleavable TcdA was reversible. These findings argue for cytosolic accumulation of the released glucosyltransferase domain of wild-type TcdA and rapid degradation of the non-cleavable TcdA. In summary, extracellular cleavage functionally inactivates TcdA (and TcdB), whereas intracellular autoproteolytic cleavage is not essential for function of TcdA but defines its potency.

Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmix.

Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1alpha (MIP-1alpha) and monocyte-chemoattractant protein-1beta (MIP-1beta) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration.

Prevention of Clostridium sordellii lethal toxin-induced apoptotic cell death by tauroursodeoxycholic acid.

Virulent strains of Clostridium sordellii cause gangrenous myonecrosis in humans. The released lethal toxin (TcsL) and hemorrhagic toxin (TcsH) are regarded as the major virulence factors. TcsL inactivates low molecular weight GTP-binding proteins of the Rho/Ras subfamilies by monoglucosylation. In cultured cell lines, glucosylation, i.e., inactivation of Rho/Ras proteins, results in actin reorganization ("cytopathic effect") and apoptotic cell death ("cytotoxic effect"). Apoptotic cell death induced by TcsL is suggested to be based on inhibition of the phosphoinositide 3-kinase (PI3K)/Akt-survival pathway. In this study, we analyze the critical role of PI3K/Akt signaling in TcsL-induced apoptosis using the antiapoptotic bile acid tauroursodeoxycholic acid (TUDCA) as the pharmacological tool. TUDCA preserved the TcsL-induced decrease of the cellular level of phospho-Akt, suggesting that TUDCA activated PI3K/Akt signaling downstream of inhibited Ras signaling. TcsL-induced apoptosis was prevented by TUDCA treatment. The antiapoptotic effect of TUDCA was abolished by the PI3K inhibitor LY294002 and the Akt inhibitor, showing that the antiapoptotic effect depends on PI3K/Akt signaling. Inhibition of Ras/Rho signaling by TcsL resulted in activation of p38 MAP kinase. Inhibition of p38 MAP kinase by SB203580 protected cells from TcsL-induced apoptosis. TUDCA induced activation of p38 MAP kinase as well, an aspect of the TUDCA effects that most likely did not contribute to its antiapoptotic activity. Due to its antiapoptotic activity, TUDCA is under investigation for its potential application as a therapeutic modulator of apoptosis-related diseases. TUDCA may represent a new concept for the treatment of disease associated with toxigenic C. sordellii.

Serine-71 phosphorylation of Rac1/Cdc42 diminishes the pathogenic effect of Clostridium difficile toxin A.

Clostridium difficile toxin A and B (TcdA/TcdB) are glucosyltransferases that glucosylate GTPases of the Rho family. The epidermal growth factor (EGF) positively modulates C. difficile toxin-induced disturbance of the intestinal barrier function by an unknown mechanism. We found that EGF-treated CaCo-2 monolayers were less susceptible to TcdA-catalysed glucosylation of Rac1 but not of RhoA, which correlated with phosphorylation of Rac1 at Ser-71. Phospho-Rac1/phospho-Cdc42 (Ser-71) still bound to the PAK-CRIB domain indicating an active state. A more detailed characterization of phospho-Rac1 was performed using the phosphomimetic mutant Rac1 S71E. Ectopic expression of Rac1 S71E induced a specific phenotype of cells showing an increase in filopodial structures that were also induced by EGF. Rac1 S71E (and Cdc42 S71E) but not Rac1 S71A was at least fivefold weaker substrate for TcdA-catalysed glucosylation compared with wild type Rac1. The protective effect was checked in transfection experiments where Rac1 S71E and, to a lesser extent, Cdc42 S71E reduced the TcdA-induced cytopathic effect. Thus, Ser-71 phosphorylation of Rac1 might be interesting for modulation of microbial pathogenesis where Rho GTPases, especially Rac1 and Cdc42, are involved. In addition, this is the first description of a specific functional outcome of Rac1 phosphorylation at Ser-71.

Killing of rat basophilic leukemia cells by lethal toxin from Clostridium sordellii: critical role of phosphatidylinositide 3'-OH kinase/Akt signaling.

Clostridium sordellii lethal toxin (TcsL) belongs to the family of clostridial glucosylating toxins. TcsL exhibits glucosyltransferase activity to inactivate Rho and Ras proteins. On cultured cells, TcsL causes actin reorganization ("cytopathic effect") and apoptotic cell death ("cytotoxic effect"). This study is based on the concept that the cytotoxic effects of TcsL depend on the glucosylation of critical substrate proteins rather than on the glucosyltransferase activity per se. The cytotoxic effects of TcsL depend on the glucosyltransferase activity of TcsL, as neither chemically inactivated TcsL nor a glucosyltransferase-deficient mutant version of TcsL caused it. The TcsL homologous toxin B from Clostridium difficile serotype F strain 1470 (TcdBF) also failed to cause cytotoxic effects. Correlation of the toxins' respective protein substrate specificities highlighted (H/K/N)Ras as critical substrate proteins for the cytotoxic effects. (H/K/N)Ras are critical upstream regulators of phosphatidylinositide 3'-OH kinase (PI3K)/Akt survival signaling. Tauroursodeoxycholic acid (TUDCA) classified to activate PI3K/Akt signaling downstream of apoptosis-inducing stimuli prevented the cytotoxic effects of TcsL. In conclusion, (H/K/N)Ras glucosylation and subsequent inhibition of PI3K/Akt signaling are critical for the cytotoxic effects of TcsL.

Adenylate cyclase toxin subverts phagocyte function by RhoA inhibition and unproductive ruffling.

Adenylate cyclase toxin (CyaA or ACT) is a key virulence factor of pathogenic Bordetellae. It penetrates phagocytes expressing the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1 or CR3) and paralyzes their bactericidal capacities by uncontrolled conversion of ATP into a key signaling molecule, cAMP. Using pull-down activity assays and transfections with mutant Rho family GTPases, we show that cAMP signaling of CyaA causes transient and selective inactivation of RhoA in mouse macrophages in the absence of detectable activation of Rac1, Rac2, or RhoG. This CyaA/cAMP-induced drop of RhoA activity yielded dephosphorylation of the actin filament severing protein cofilin and massive actin cytoskeleton rearrangements, which were paralleled by rapidly manifested macrophage ruffling and a rapid and unexpected loss of macropinocytic fluid phase uptake. As shown in this study for the first time, CyaA/cAMP signaling further caused a rapid and near-complete block of complement-mediated phagocytosis. Induction of unproductive membrane ruffling, hence, represents a novel sophisticated mechanism of down-modulation of bactericidal activities of macrophages and a new paradigm for action of bacterial toxins that hijack host cell signaling by manipulating cellular cAMP levels.