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ABSTRACT: Clostridium difficile toxins A and B are major virulence factors responsible for induction of pseudomembranous colitis and antibiotic-associated diarrhea in men. The toxins possess a multidomain structure and only the N-terminal glucosyltransferase domain, which inactivates Rho GTPases by glucosylation, is translocated into the cytosol of target cells. Processing of the toxin occurs by autocatalytic cleavage and is activated by inositol hexakisphosphate (InsP6). Here we studied the inherent protease activity in fragments of toxin B and determined the site of toxin B that interacts with InsP6. We report that a fragment of toxin B, comprised of residues 1-955, is cleaved in the presence of InsP6. In contrast, mutants of the catalytic triad of the putative cysteine protease domain did not cleave this fragment. [3H]InsP6 bound to holotoxin B and to the fragment 1-955, but not to a fragment comprising residues 900-2366 or the glucosyltransferase domain (residues 1-544). Binding to the putative cysteine protease domain (residues 544-955) was also observed. InsP6-binding was specific and saturable. Isothermal titration calorimetry revealed a Kd value of 2.4 microm for binding of InsP6 to toxin fragment 544-955 with a stoichiometry factor of 0.86. Lysine 600 of toxin B was identified as essential amino acid for InsP6 binding and for InsP6-dependent activation of the protease activity.
Journal of Biological Chemistry 01/2009; 284(6):3389-95. · 4.77 Impact Factor
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ABSTRACT: The pathogenicity of Clostridium difficile depends on the large clostridial glucosylating toxins A and B (TcdA and TcdB). The proteins accomplish their own uptake by a modular structure comprising a catalytic and a binding/translocation domain. Based on a proteolytic processing step solely the catalytic domain reaches the cytosol. Within the cells, the glucosyltransferases inactivate small GTPases by mono-O-glucosylation. Here, a short overview is given regarding latest insights into the intramolecular processing, which is mediated by an intrinsic protease activity.
Journal of Medical Microbiology 07/2008; 57(Pt 6):690-6. · 2.50 Impact Factor
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ABSTRACT: Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human alpha-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of alpha-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B.
Inactivation of C difficile toxins by alpha-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine K(m) and median inhibitory concentration (IC(50)) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies.
Treatment of cells with human alpha-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only alpha-defensins, but not beta-defensin-1 or cathelicidin LL-37, inhibited toxin B-catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing K(m) values for uridine 5'-diphosphate-glucose up to 10-fold. The IC(50) values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 micromol/L. At high concentrations, defensins (HNP-1 > or = 2 micromol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor.
Our data indicate that toxin B interacts with high affinity with alpha-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.
Gastroenterology 06/2008; 134(7):2049-58. · 11.68 Impact Factor
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ABSTRACT: Recently the crystal structure of the catalytic domain of Clostridium difficile toxin B was solved ( Reinert, D. J., Jank, T., Aktories, K., and Schulz, G. E. (2005) J. Mol. Biol. 351, 973-981 ). On the basis of this structure, we studied the functional role of several amino acids located in the catalytic center of toxin B. Besides the (286)DXD(288) motif and Trp(102), which were shown to be necessary for Mn(2+) and UDP binding, respectively, we identified by alanine scanning Asp(270), Arg(273), Tyr(284), Asn(384), and Trp(520) as being important for enzyme activity. The amino acids Arg(455), Asp(461), Lys(463), and Glu(472) and residues of helix alpha17 (e.g. Glu(449)) of toxin B are essential for enzyme-protein substrate recognition. Introduction of helix alpha17 of toxin B into Clostridium sordellii lethal toxin inhibited modification of Ras subfamily proteins but enabled glucosylation of RhoA, indicating that helix alpha17 is involved in RhoA recognition by toxin B. The data allow the design of a model of the interaction of the glucosyltransferase domain of toxin B with its protein substrate RhoA.
Journal of Biological Chemistry 12/2007; 282(48):35222-31. · 4.77 Impact Factor
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ABSTRACT: The action of Clostridium difficile toxins A and B depends on processing and translocation of the catalytic glucosyltransferase domain into the cytosol of target cells where Rho GTPases are modified. Here we studied the processing of the toxins. Dithiothreitol and beta-mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into approximately 250/210- and approximately 63-kDa fragments. The 63-kDa fragment was identified by mass spectrometric analysis as the N-terminal glucosyltransferase domain. This cleavage was blocked by N-ethylmaleimide or iodoacetamide. Exchange of cysteine 698, histidine 653, or aspartate 587 of toxin B prevented cleavage of full-length recombinant toxin B and of an N-terminal fragment covering residues 1-955 and inhibited cytotoxicity of full-length toxin B. Dithiothreitol synergistically increased the effect of myo-inositol hexakisphosphate, which has been reported to facilitate auto-cleavage of toxin B (Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., Schild, H., and Von Eichel-Streiber, C. (2007) Nature 446, 415-419). N-Ethylmaleimide blocked auto-cleavage induced by the addition of myo-inositol hexakisphosphate, suggesting that cysteine residues are essential for the processing of clostridial glucosylating toxins. Our data indicate that clostridial glucosylating cytotoxins possess an inherent cysteine protease activity related to the cysteine protease of Vibrio cholerae RTX toxin, which is responsible for auto-cleavage of glucosylating toxins.
Journal of Biological Chemistry 09/2007; 282(35):25314-21. · 4.77 Impact Factor
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ABSTRACT: Clostridium difficile causes pseudomembranous colitis and is responsible for many cases of nosocomial antibiotic-associated diarrhea. Major virulence factors of C. difficile are the glucosylating exotoxins A and B. Both toxins enter target cells in a pH- dependent manner from endosomes by forming pores. They translocate the N-terminal catalytic domains into the cytosol of host cells and inactivate Rho guanosine triphosphatases by glucosylation. The crystal structure of the catalytic domain of toxin B was solved in a complex with uridine diphosphate, glucose, and manganese ion, exhibiting a folding of type A family glycosyltransferases. Crystallization of fragments of the C-terminus of toxin A, which is characterized by polypeptide repeats, revealed a solenoid-like structure often found in bacterial cell surface proteins. These studies, which provide new insights into structure, uptake, and function of the family of clostridial glucosylating toxins, are reviewed.
Glycobiology 05/2007; 17(4):15R-22R. · 3.58 Impact Factor
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ABSTRACT: Mast cells (MCs) play central roles for the onset and development of immediate-type and inflammatory allergic reactions. Since the inverse relationship between atopic disorders and diabetes mellitus has been observed in animals and humans, we investigated the effects of insulin (Ins) on MC signaling and biological function.
In bone marrow-derived MCs (BMMCs) from wild-type as well as SHIP-deficient mice Ins as well as insulin-like growth factor-1 (IGF-I)-triggered intracellular signaling events and MC effector functions were studied.
We found that the addition of either Ins or IGF-1 to BMMCs triggers the phosphorylation of protein kinase B (PKB) and p38 kinase but not extracellular signal-regulated kinase (Erk). We also found that Ins/IGF-1 stimulates the tyrosine phosphorylation of SHIP1 and, in keeping with this, Ins/IGF-1-induced PKB phosphorylation is higher in SHIP1-/- BMMCs and is inhibited in SHIP+/+ as well as SHIP1-/- BMMCs with inhibitors of phosphatidylinositol-3-kinase (PI3K). Ins/IGF-1, like antigen (Ag), also stimulates the Rac-dependent activation of PAK as well as the production of hydrogen peroxide (H2O2). To elucidate the role of Ins and IGF-1 in MC biology, we studied their effects on Ag-mediated degranulation and MC survival. Although both only slightly enhanced Ag-mediated degranulation, they significantly promoted MC survival in the absence of IL-3 in a PI3K-dependent manner.
The promotion of BMMC survival by induction of Ins/IGF-1 signaling may, in part, be responsible for the inverse correlation observed between atopic disorders and diabetes mellitus.
Experimental Hematology 12/2006; 34(11):1532-41. · 2.90 Impact Factor
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ABSTRACT: Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.
Journal of Biological Chemistry 08/2006; 281(28):19527-35. · 4.77 Impact Factor
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ABSTRACT: The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of 86Rb+-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited 86Rb+ efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.
Journal of Biological Chemistry 05/2006; 281(16):10808-15. · 4.77 Impact Factor
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ABSTRACT: The large cytotoxins of Clostridia species glycosylate and thereby inactivate small GTPases of the Rho family. Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin use UDP-glucose as the donor for glucosylation of Rho/Ras GTPases. In contrast, alpha-toxin from Clostridium novyi N-acetylglucosaminylates Rho GTPases by using UDP-N-acetylglucosamine as a donor substrate. Based on the crystal structure of C. difficile toxin B, we studied the sugar donor specificity of the toxins by site-directed mutagenesis. The changing of Ile-383 and Gln-385 in toxin B to serine and alanine, respectively, largely increased the acceptance of UDP-N-acetylglucosamine as a sugar donor for modification of RhoA. The K(m) value was reduced from 960 to 26 mum for the double mutant. Accordingly, the potential of the double mutant of toxin B to hydrolyze UDP-N-acetylglucosamine was higher than that for UDP-glucose. The changing of Ile-383 and Gln-385 in the lethal toxin of C. sordellii allowed modification of Ras in the presence of UDP-N-acetyl-glucosamine and reduced the acceptance of UDP-glucose as a donor for glycosylation. Vice versa, the changing of the equivalent residues in C. novyi alpha-toxin from Ser-385 and Ala-387 to isoleucine and glutamine, respectively, reversed the donor specificity of the toxin from UDP-N-acetylglucosamine to UDP-glucose. These data demonstrate that two amino acid residues are crucial for the co-substrate specificity of clostridial glycosylating toxins.
Journal of Biological Chemistry 12/2005; 280(45):37833-8. · 4.77 Impact Factor
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Torsten Giesemann,
Günter Schwarz,
Ralph Nawrotzki,
Kerstin Berhörster,
Martin Rothkegel,
Kathrin Schlüter,
Nils Schrader,
Hermann Schindelin,
Ralf R Mendel,
Joachim Kirsch,
Brigitte M Jockusch
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ABSTRACT: Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.
Journal of Neuroscience 10/2003; 23(23):8330-9. · 7.11 Impact Factor
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ABSTRACT: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of α-motoneurons in the spinal
cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven
exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to
bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with
SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding
depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue.
We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons.
In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN
in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although
SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the
SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific
effects of SMN mutations.
Journal of Biological Chemistry 12/1999; 274(53):37908-37914. · 4.77 Impact Factor
