Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L.

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.     

Calcium and calmodulin in cellular intoxication with Clostridium difficile toxin B

In cultured human lung fibroblasts treated with Clostridium difficile toxin B, the development of the cytopathogenic effect was inhibited by the proton ionophore monensin but was not affected by some other ionophores. The calcium channel blockers verapamil and LaCl3 protected the cells against intoxication, as did the calmodulin antagonists trifluoperazine, amitriptyline, R 24571, and dansylcadaverine. Since these agents could not prevent intoxication when added after the toxin internalization was completed, we suggest that calmodulin and uptake of extracellular calcium are needed for the internalization but not for the cytosolic action of the toxin.     

The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.     

N-acetylcysteine protects epithelial cells against the oxidative imbalance due to Clostridium difficile toxins.

Toxins A and B from the anaerobic bacterium Clostridium difficile are the causative agents of the antibiotic-associated pseudomembraneous colitis. At the subcellular level, they inhibit the Rho family GTPases, thus causing alterations of the actin cytoskeleton. The cytoskeletal integrity is also controlled by the redox state of cells. Therefore, we have evaluated whether an oxidative imbalance could be involved in the toxin-induced cytopathic effects. Our results indicate that both toxins induce oxidative stress with a significant depletion of protein SH-groups. These responses and the cytoskeleton-dependent cell retraction and rounding are significantly counteracted by N-acetylcysteine but not by alpha-tocopherol. Our study provides the first evidence that the thiol supplier N-acetylcysteine impairs the cellular intoxication by acting on the cytoskeleton integrity. This also suggests a possible beneficial role for this drug during therapeutic intervention.     

Inhibition and redistribution of NHE3, the apical Na+/H+ exchanger, by Clostridium difficile toxin B

NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.     

Polymeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.     

重组艰难梭菌毒素B对小鼠结肠癌CT26细胞的诱导凋亡作用

本文探讨了重组艰难梭菌毒素B(rTcd B)对小鼠结肠癌CT26细胞的诱导凋亡作用。采用不同浓度rTcd B处理CT26细胞, 通过MTT法检测细胞增殖抑制率; 比色法测定Caspase 3活性; 细胞形态学和流式细胞技术检测细胞凋亡。结果表明, rTcd B显著抑制了CT26细胞的增殖, 并呈时间?剂量依赖性; Caspase 3活性在处理6 h后显著升高, 至18 h达到最大值, 与对照组相比差异显著, 具有统计学意义(P<0.05); 荧光显微镜观察到典型细胞凋亡形态学变化, 细胞膜内侧的磷脂酰丝氨酸(PS)异位到了膜外侧, 细胞膜呈明亮的绿色荧光; 通过流式细胞仪检测结果表明, 细胞凋亡率呈时间?剂量依赖性增加。实验结果表明, 重组艰难梭菌毒素B能够诱导小鼠结肠癌CT26细胞凋亡。     

Effect of the cytotoxin of Clostridium difficile on cultured hepatoma cells

Clostridium difficile is the major etiologic agent of human pseudomembranous colitis. It produces two toxins: an enterotoxin and a cytotoxin. In cultured hepatoma cells, at very low doses, the cytotoxin inhibits the incorporation of precursors into biological macromolecules. Protein synthesis is more affected than RNA and DNA synthesis. The toxin also induces severe alterations of the cell morphology consisting in damages to the cytoskeleton and to the cell shape.     

Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture

One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [³⁵S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.     

cAMP-induced cytoskeleton rearrangement increases calcium transients through the enhancement of capacitative calcium entry

In this report we investigated the correlation between cell morphology and regulation of cytosolic calcium homeostasis. Type I astrocytes were differentiated to stellate process-bearing cells by a 100-min exposure to cAMP. Differentiation of cortical astrocytes increased the magnitude and duration of calcium transients elicited by phospholipase C-activating agents as measured by single cell Fura-2-based imaging. Calcium imaging showed differences in the spatial pattern of the response. In both differentiated and the control cells, the response originated in the periphery and gradually extended into the center of the cell. However, the elevation of cytosolic calcium concentration ([Ca(2+)](i)) was particularly evident within the processes and adjacent to the inner cell membrane of the differentiated astrocytes. In addition, differentiation significantly prolonged the duration of the [Ca(2+)](i) elevation. Potentiation of the calcium transients was mimicked by forskolin-induced differentiation and abolished by a specific protein kinase-A blocker. Conversely, the enhancement of the calcium transients was not mimicked by brief exposure to cAMP not causing morphological differentiation, and in PC12 cells that did not undergo morphological changes after 100 min of cAMP treatment. Impairing cAMP-induced cytoskeleton re-organization, by means of cytochalasin D and nocodazole, prevented the potentiation of the calcium transients in cAMP-treated astrocytes. Phospholipase C activity and sensitivity to inositol (1,4,5)-trisphosphate were not involved in the enhancement of the calcium responses. Also, potentiation of the calcium transients was dependent on extracellular calcium. Calcium storage and thapsigargin-depletable intracellular calcium reservoirs were analogously not increased in differentiated astrocytes. Rearrangement of the cell shape also caused a condensation of the endoplasmic reticulum and altered the spatial relationship between the endoplasmic reticulum and the cell membrane. In conclusion, morphological rearrangements of type I astrocytes increase the magnitude and the duration of agonist-induced calcium transients via enhancement of capacitative calcium entry and is associated with a spatial reorganization of the relationship between cell membrane and the endoplasmic reticulum structures.     

Microfilament-disrupting Clostridium difficile toxin B causes multinucleation of transformed cells but does not block capping of membrane Ig

The effects of Clostridium difficile toxin B on some actin-dependent cellular functions were studied. A three-day incubation of intoxicated B-lymphocytes and transformed 3T3 fibroblasts resulted in dose-dependent multinucleation. Using vimentin-negative Daudi cells we showed that this effect of toxin B does not involve vimentin. As DNA and protein syntheses are not impaired in the cells used, the results suggest that toxin B has an effect on the actin-containing contractile ring during mitosis, in a manner similar to that of the microfilament-disrupting agent cytochalasin B. Toxin B is the first bacterial toxin shown to have this effect. It was also found that the capping of surface IgM on B-lymphocytes was not inhibited by toxin B, whereas cytochalasin B did inhibit capping. These results suggest that capping is dependent on a specific membrane-associated actin structure, which is not affected by toxin B.     

An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival

Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.     

Super toxins from a super bug: structure and function of Clostridium difficile toxins

Clostridium difficile, a highly infectious bacterium, is the leading cause of antibiotic-associated pseudomembranous colitis. In 2009, the number of death certificates mentioning C. difficile infection in the U.K. was estimated at 3933 with 44% of certificates recording infection as the underlying cause of death. A number of virulence factors facilitate its pathogenicity, among which are two potent exotoxins; Toxins A and B. Both are large monoglucosyltransferases that catalyse the glucosylation, and hence inactivation, of Rho-GTPases (small regulatory proteins of the eukaryote actin cell cytoskeleton), leading to disorganization of the cytoskeleton and cell death. The roles of Toxins A and B in the context of C. difficile infection is unknown. In addition to these exotoxins, some strains of C. difficile produce an unrelated ADP-ribosylating binary toxin. This toxin consists of two independently produced components: an enzymatic component (CDTa) and the other, the transport component (CDTb) which facilitates translocation of CDTa into target cells. CDTa irreversibly ADP-ribosylates G-actin in target cells, which disrupts the F-actin:G-actin equilibrium leading to cell rounding and cell death. In the present review we provide a summary of the current structural understanding of these toxins and discuss how it may be used to identify potential targets for specific drug design.     

CD14 expression by human mononuclear phagocytes is modulated by Clostridium difficile toxin B

Toxin B, an exotoxin produced by the anaerobic Gram-positive bacteria Clostridium difficile, is responsible for pseudomembranous colitis in humans. It deeply modifies morphology of cultured cells and enhances their membrane surface area, which suggests a possible alteration of membrane receptor distribution. Since toxin B and bacterial lipopolysaccharide can act synergistically on TNF-alpha production by mononuclear phagocytes, the effect of toxin B on CD14 expression was investigated using flow cytometric analysis. It was shown that monocytes overexpressed CD14 after 5 h of treatment with toxin B. In contrast, after 24 h of treatment, the percentage of CD14 monocytes decreased, although, most frequently, the remaining positive cells expressed high levels of CD14 compared with untreated cells. Macrophages treated for 5 h with toxin B overexpressed CD14, but this effect persisted for at least 24 h. Both the percentage of positive macrophages and the mean level of CD14 per cell were increased. Thus toxin B can modulate expression of CD14 and its modulation depends on the differentiation status and maybe on the activation state, since some individual variations were observed in monocyte response to toxin.     

Differential effects of calcium deprivation on the cytoskeleton of non-tumorigenic and tumorigenic rat liver cells in culture

Normal rat liver T51B epithelial cells and Morris no. 7795 hepatoma cells growing exponentially were exposed for 24 h to standard medium containing low (0.02 mM) calcium, a concentration which drastically reduces the proliferation of normal but not tumour cells. Cell surface morphology was examined by scanning electron microscopy (SEM); and the distribution and organization of microtubules, cytokeratin and vimentin filaments, and microfilaments were analysed by indirect immunofluorescence microscopy using specific antibodies. Calcium deprivation caused the loss of intercellular cohesion in both cell types and the appearance of some microvilli and blebs, particularly on tumour cells. However, marked differential (normal vs tumour cells) effects on the organizational integrity of the cytoskeleton fibrillar network were observed. Extracellular calcium deprivation led to a particular rearrangement of microtubules, and a perinuclear accumulation of cytokeratin and vimentin filaments in normal, but not in tumour cells. A massive concentration of actin-containing microfilaments was observed in the cell periphery and blebs of hepatoma cells. In the light of the possible involvement of calcium in controlling cytoskeleton assembly, the differing cytoskeletal changes of the two cell types may be linked to their diffferent proliferative capabilities in low-calcium medium.     

Distinct effects of calcium- and cyclic AMP-enhancing factors on cytoskeletal synthesis and assembly in mouse osteoblastic cells.

Previous studies have indicated that the effects of parathyroid hormone (PTH) on osteoblastic function involve alteration of cytoskeletal assembly. We have reported that after a transitory cell retraction, PTH induces respreading with stimulation of actin, vimentin and tubulins synthesis in mouse bone cells and that this effect is not mediated by cAMP. In order to further elucidate the role of intracellular cAMP and calcium on PTH action on bone cell shape and cytoskeleton we have compared the effects of calcium- and cAMP-enhancing factors on actin, tubulin and vimentin synthesis in relation with mouse bone cell morphology, DNA synthesis and alkaline phosphatase activity as a marker of differentiation. Confluent mouse osteoblastic cells were treated with 0.1 mM isobutylmethylxanthine (IBMX) for 24 h. This treatment caused an increase in the levels of cytoskeletal subunits associated with an elevation of cAMP. Under these conditions, PTH (20 nM) and forskolin (0.1 microM) produced persistent cytoplasmic retraction. PTH and forskolin treatment in presence of IBMX (24 h) induced inhibitory effects on actin and tubulin synthesis evaluated by [35S]methionine incorporation into cytoskeletal proteins identified on two-dimensional gel electrophoresis. Under these culture conditions PTH and forskolin also caused disassembly of microfilament and microtubules as shown by the marked reduction in Triton X soluble-actin and alpha- and beta-tubulins. In contrast, incubation of mouse bone cells with 1 microM calcium ionophore A23187 (24 h) resulted in increased monomeric and polymeric forms of actin and tubulin while not affecting intracellular cAMP. Alkaline phosphatase activity was increased in all conditions while DNA synthesis evaluated by [3H]thymidine incorporation into DNA was stimulated by PTH combined with forskolin and inhibited by the calcium ionophore. These data indicate that persistent elevation of cAMP levels induced by PTH and forskolin with IBMX cause cell retraction with actin and tubulin disassembly whereas rising cell calcium induces cytoskeletal protein assembly and synthesis in mouse osteoblasts. The results point to a distinct involvement of calcium and cAMP in both cytoskeletal assembly and DNA synthesis in mouse bone cells.     

Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT.

Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now. It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation. Here, we describe some effects of toxin A on the leukemic T cell line JURKAT. Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin. However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent. After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter. These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment. These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation. The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation.     

Thyroid Hormone-Induced Maturation of Astrocytes Is Associated with the Expression of New Variants of Vimentin and Their Phosphorylation

The role of thyroid hormone (TH)-induced vimentin expression in promoting the maturation of astrocytes has been investigated. Comparative immunocytochemical staining with vimentin antibody demonstrated distinctly different patterns of expression of vimentin during the progressive maturation of astrocytes of normal and TH-deficient astrocyte cultures. [35S]Methionine labeling of cells showed a significant decline in the level of vimentin in the hypothyroid cultures at all ages from day 5 to 20. Western blot analysis suggested that the maturation of normal astrocytes was associated with the appearance of several acidic and basic variants of vimentin, whose expression was significantly delayed or reduced in the TH-deficient astrocytes. In normal astrocyte cultures, two acidic variants of vimentin, one of similar molecular weight and the other of lower molecular weight, were found to be phosphorylated during the final transformation of the epithelioid form into the mature stellate form. None of these phosphorylated forms was evident in the TH-deficient astrocytes. This was further confirmed by negative immunocytochemical staining of 5- to 18-day-old cultures of TH-deficient astrocytes with antibody TM71, specific to phosphorylated vimentin, compared with age-matched normal astrocytes, which displayed consistent positive staining. The overall results indicate that TH-induced expression of phosphorylated forms of vimentin may be essential for the intracellular organization of the cytoskeleton in a way conducive to the morphological maturation of astrocytes.     

Phorbol Myristate Acetate and 8-Bromo-Cyclic AMP-Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)