GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42.

Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.

Inhibition of small G proteins of the rho family by statins or clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II.

In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG-CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho-proteins directly. Human A549/8- and DLD-1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1 - 100 microM) or TcdB (0.01-10 ng ml(-1)). Then NOS II expression was induced by cytokines. NOS II mRNA was measured after 4 - 8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h. Statins and TcdB markedly increased cytokine-induced NOS II mRNA expression and NO production. Statin-mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate. It was only slightly reduced by farnesylpyrophosphate. Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction. In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine-induced NOS II promoter activity. In contrast, statins had a considerable superinducing effect in DLD-1 cells stably transfected with pNOS2(16)Luc. In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine-induced NOS II expression via inhibition of small G proteins of the Rho family. This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability.

Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and rho GTPases in NIH 3T3 fibroblasts involvement of both phosphatidylinositol-4-phosphate 5-kinase and phospholipase C.

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4, 5)P2-producing enzyme, PtdIns4P 5-kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30-50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1. Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C.

Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864--implications for transcription, expression and enzymatic activity of toxins A and B.

The pathogenicity locus (PaLoc) of Clostridium difficile isolate 8864 was investigated to locate genetic rearrangements that would explain the exceptional pathogenicity of this particular isolate. Two major changes were defined: an insertion of 1.1 kb between the two genes tcdA and tcdE, coding for the enterotoxin and an accessory protein of unknown function, respectively, and a deletion of 5.9 kb encompassing the 3' ends of tcdA and tcdC. Transcription of the tcdA-E genes is severely affected by both rearrangements, explaining the demonstrated complete lack of TcdA polypeptide. We present a model of coordinate, growth-related transcription of the tcdA-E genes that confirms our previous findings in strain 10463. Recombinant TcdA-8864 had UDP-glucose-glucosyltransferase activity, proving that the N-terminal 698 amino acids of the polypeptide represent the catalytic domain. However, this truncated TcdA molecule lacks a ligand and translocation domain. To assess the catalytic domain of TcdB-8864, the sequence of the 5' end of its gene was determined. TcdB-8864 shows high homology to TcdB-1470 but lower homology to TcdB-10463 within this domain. This fits well with the altered glucosylation specificity of TcdB-8864 (Rac1, Rap2 and Ra1). Having defined the variations of transcription, expression and enzymatic activity of toxins A and B, implications for the pathogenic potential of strain 8864 are discussed.

Rho protein inhibition blocks protein kinase C translocation and activation.

Small GTP-binding proteins of the Ras and Rho family participate in various important signalling pathways. Large clostridial cytotoxins inactivate GTPases by UDP-glucosylation. Using Clostridium difficile toxin B-10463 (TcdB) for inactivation of Rho proteins (RhoA/Rac/Cdc42) and Clostridium sordellii lethal toxin-1522 (TcsL) for inactivation of Ras-proteins (Ras/Rac/Ral, Rap) the role of these GTPases in protein kinase C (PKC) stimulation was studied. Phorbol-myristate-acetate (PMA) induced a rapid PKC translocation to and activation in the particulate cell fraction as determined by PKC-activity measurements and Western blots for PKC alpha. These effects were blocked by TcdB inhibiting Rho proteins in endothelial cells, but not in TcsL-treated cells (i.e., cells without Ras activity), suggesting that Rho GTPases (RhoA and/or Cdc42) are the most likely GTP-binding proteins responsible for PKC activation. The Rho requirement for PKC activation/translocation was also verified for human epithelial cells and for lipopolysaccharide-stimulated endothelial cells. In summary, the data presented indicate that Rho protein inhibition blocked PKC translocation/activation in endothelial and epithelial cells.

The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality.

We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Le(x)-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Le(x) as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.

Delineation of the catalytic domain of Clostridium difficile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment.

In an attempt to directly approach the postulated toxic domain of Clostridium difficile's TcdB-10463, eight subclones of different size and locations in the N-terminal third of the toxin were generated. Expression of these toxin fragments was checked in Western blots and the enzymatic activity of the expressed proteins was analyzed by glucosylating Ras related small GTP-binding proteins. Two polypeptides of 875 aa (TcdBc1-3) and 557 aa (TcdBc1-H) glucosylated their targets Rho, Rac and Cdc42 with the same activity and specificity as the holotoxin. In comparison 516 aa (TcdBc1-N) and 467 aa (TcdBc1-A) protein fragments exhibited highly reduced activity, while Tcdc1 and TcdB2-3 (aa 1-243 and 244-890, respectively) were enzymatically inactive. Our results indicate that all structures involved in the catalysis are located at several different sites within the 557 aa fully active fragment. The shortest enzymatically still active protein covers aa 1-467 and obviously fulfils all minimal requirements for glucosylation. The data support the postulated three domain model of 'large clostridial cytotoxins'.

Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.

Lethal toxin (LT) from Clostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serum-starved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.

Inhibition of Fc epsilon-RI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase)

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.

Sequencing and analysis of the gene encoding the alpha-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile.

A library of total Clostridium novyi DNA was established and screened for the alpha-toxin gene (tcn alpha) by hybridization with oligonucleotides derived from a partial N-terminal sequence and by using specific antisera. Overlapping subgenic tcn alpha fragments were isolated and subsequently the total sequence of tcn alpha was determined. The 6534 nucleotide open reading frame encodes a polypeptide of M(r) 250,166 and pI 5.9. The N-terminal alpha-toxin (Tcn alpha) sequence MLITREQLMKIASIP determined by Edman degradation confirmed the identity of the reading frame and the assignment of the translation start point. The toxin is not modified posttranslationally at its N-terminus nor does it consist of different subunits. Overall the amino acid sequence shows 48% homology between the Tcn alpha and both toxins A (TcdA) and B (TcdB) of Clostridium difficile. The C-terminal 382 residues of Tcn alpha constitute a repetitive domain similar to those reported for TcdA and TcdB of C. difficile. The individual repeat motifs of these three toxins consist of oligopeptides some 19-52 amino acids in length, arranged in four to five different groups. Genetic, biochemical and pharmacological data thus confirm that the three toxins belong to one subgroup, designated large clostridial cytotoxins (LCT). Further definition of their structure and detailed molecular action should allow the LCTs to be used tools for the analysis of microfilament assembly and function.

The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.

Glucosylation of Rho proteins by Clostridium difficile toxin B.

Toxin A and B, the major virulence factors of Clostridium difficile, are the causative agents of antibiotic-associated pseudomembranous colitis. In cultured cell lines their potent cytotoxicity results from their ability to induce disaggregation of the microfilament cytoskeleton. Toxin B acts on the low-molecular-mass GTPase RhoA, which is involved in the regulation of the actin cytoskeleton. We report here that toxin B catalyses the incorporation of up to one mole of glucose per mole of RhoA at the amino acid threonine at position 37. The modification was identified and localized by tandem electrospray mass spectrometry. UDP-glucose selectively serves as cosubstrate for the monoglucosylation reaction catalysed by toxin B. Microinjection of RhoA previously glucosylated by toxin B into monolayer cells caused disaggregation of actin filaments, indicating a dominant-negative activity of glucosylated RhoA.

The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile.

Enterotoxin A is one of the major virulence factors of Clostridium difficile, and the causative agent of antibiotic-associated pseudomembranous colitis. In cell culture (NIH-3T3, rat basophilic leukemia cells) toxin A inhibits Clostridium botulinum ADP-ribosyltransferase C3 (C3)-catalyzed ADP-ribosylation of the low molecular mass GTP-binding Rho proteins. Rho participates in the regulation of the microfilament cytoskeleton. Decrease in ADP-ribosylation of Rho occurs in a time- and concentration-dependent manner and precedes the toxin A-induced destruction of the actin cytoskeleton. Action of toxin A is not due to proteolytical degradation of Rho or to an inherent ADP-ribosyltransferase activity of toxin A. Toxin A-induced decrease in ADP-ribosylation is observed also in cell lysates and with recombinant RhoA protein. A heat stable low molecular mass cytosolic factor is essential for the toxin effect on Rho. Thus, the enterotoxin (toxin A) resembles the effects of the C. difficile cytotoxin (toxin B) on Rho proteins (Just, I., G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbath, and C. Von Eichel-Streiber. 1994. J. Biol. Chem. 269:10706-10712). The data indicate that despite different in vivo effects, toxin A and toxin B act on the same cellular target protein Rho to elicit their toxic effects.

Clostridium difficile toxin B acts on the GTP-binding protein Rho.

Clostridium difficile toxin B exhibits cytotoxic activity that is characterized by the disruption of the microfilamental cytoskeleton. Here we studied whether the GTP-binding Rho protein, which reportedly participates in the regulation of the actin cytoskeleton, is involved in the toxin action. Toxin B treatment of Chinese hamster ovary cells reveals a time- and concentration-dependent decrease in the ADP-ribosylation of Rho by Clostridium botulinum C3 exoenzyme in the cell lysate. Disruption of the microfilament system induced by C. botulinum C2 toxin or cytochalasin D does not cause impaired ADP-ribosylation of Rho. Toxin B exhibits its effects on Rho not only in intact cells but also when added to cell lysates. Besides endogenous Rho, RhoA-glutathione S-transferase (Rho-GST) fusion protein added to cell lysate showed decreased ADP-ribosylation after toxin B treatment. Immunoblot analysis reveals identical amounts of Rho-GST and no change in molecular mass after toxin B treatment compared with controls. ADP-ribosylation of Rho-GST purified from toxin B-treated cell lysate is inhibited, indicating a modification of Rho itself. Finally, transfection of rhoA DNA under the control of a strong promoter into cells protects them from the activity of toxin B. Altogether, the data indicate that C. difficile toxin B acts directly or indirectly on Rho proteins to inhibit ADP-ribosylation and suggest that the cytotoxic effect of toxin B involves Rho.

Comparative sequence analysis of the Clostridium difficile toxins A and B.

The six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCd13 cover the tox locus of Clostridium difficile VPI 10463. This region of 19 kb of chromosomal DNA contains four open reading frames including the complete toxB and toxA genes. The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera. A special feature of ToxA and ToxB is their repetitive C-termini. We define herein 19 individual CROPs (combined repetitive oligopeptides of 20-50 aa length) in the ToxB C-terminus, which are separable into five homologous groups. Comparison of the aa sequences of the N-terminal two-thirds of ToxA and ToxB revealed three marked structures, a cluster of 172 hydrophobic, highly conserved aa in the centre of both toxins, a sequence of 120 residues with an accumulation of highly conserved arginine, cysteine, histidine, methionine, and tryptophan residues, and a stretch of 248 less conserved aa. The probable function of these domains is discussed. Structural and functional homologies of ToxA and ToxB indicate that both genes have a common ancestor and may have evolved by gene duplication, with subsequent recombination and mutation, as has been reported for streptococcal glucosyltransferases (Gtf).

A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C. difficile toxin B and C. sordellii lethal toxin.

The three clostridial cytotoxins, i.e. alpha-toxin of C. novyi (Tox alpha-nov), toxin B of C. difficile (ToxB-dif) and lethal toxin of C. sordellii (LT-sor) consist of single peptide chains of about 200,000 (Tox alpha-nov), 250,000 (LT-sor) and 275,000 (ToxB-dif) mol. wts. ToxB-dif and LT-sor but not Tox alpha-nov cross-reacted with rabbit polyclonal antibodies. Toxicity upon i.v. injection in mice was similar (LD50, 100 hr, 50-200 ng/kg) and was characterized by a slowly developing fluid loss into the interstitial space. When injected into the rat paw the toxins caused a delayed local edema lasting for days. In vitro the three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ToxB-dif and Tox alpha-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. The toxins inhibited uridine incorporation into endothelial or chicken embryonic cells whereas T 84 cells responded by an about 10-fold increase of uridine incorporation. Neither toxin ADP-ribosylated actin. The similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the microfilament system.

Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B.

High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5' and 3' end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA, toxB has a size of 6.9 kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5' end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open reading frame. The N-terminal 203 amino acids of ToxB were compared with the N-terminal amino acids of the enterotoxin A (ToxA). A homology of 64% of the residues was detected, which proves the relatedness of ToxA and ToxB of C. difficile.