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C Montecucco

Publications and source records attributed to C Montecucco.

At least 73 records · Page 4Linked to original sources
PubMed1999

Recombinant and truncated tetanus neurotoxin light chain: cloning, expression, purification, and proteolytic activity.

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.

Cloning, Molecular↗
PubMed1999

Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation.

The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.

Bacterial Proteins↗
PubMed1999

Anthrax lethal factor cleaves the N-terminus of MAPKKS and induces tyrosine/threonine phosphorylation of MAPKS in cultured macrophages

The lethal toxin (LeTx) of Bacillus anthracis is the major virulence factor responsible for the death of infected animals and for cytolysis of cultured macrophages. Its catalytic component, LF, contains the characteristic zinc-binding motif of metalloproteases, it binds zinc and indirect evidence suggests that this hydrolytic activity is essential for LeTx cytotoxicity (Limpel et al. 1994; Kochi et al. 1994). To identify substrates of LF, we have used the yeast two-hybrid system, employing an LF inactive mutant as bait. This approach has led to the identification of the MAP kinase kinases (MAPKKs) Mek1 and Mek2 as proteins capable of specific interaction with LF. LF cleaves Mek1 and Mek2 within their N-terminus in vitro and in vivo, hydrolysing a Pro8-Ile9 and a Pro10-Arg11 peptide bond in Mek1 and Mek2, respectively (Vitale et al. 1998), similarly to that found with a different approach by Duesbery et al. (1998). The removal of the amino terminus of MAPKKs eliminates the 'docking site' involved in the specific interaction with MAPKs and interferes with the phospho-activation of the MAPKs ERK1 and ERK2, which become phosphorylated in cultured macrophages following toxin challenge. We are currently investigating the relevance of MAPKKs cleavage for LeTx cytotoxicity and the consequences for the activity of the MAP pathway.

Journal Article↗
PubMed1999

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.

Amino Acid Sequence↗
PubMed1999

Towards deciphering the Helicobacter pylori cytotoxin.

VacA, the major exotoxin produced by Helicobacter pylori, is composed of identical 87 kDa monomers that assemble into flower-shaped oligomers. The monomers can be proteolytically cleaved into two moieties, one of 37 and the other of 58 kDa, named P37 and P58 respectively. The most studied property of VacA is the alteration of intracellular vesicular trafficking in eukaryotic cells leading to the formation of large vacuoles containing markers of late endosomes and lysosomes. However, VacA also causes a reduction in transepithelial electrical resistance in polarized monolayers and forms ion channels in lipid bilayers. The ability to induce vacuoles is localized mostly but not entirely in P37, whereas P58 is mostly involved in cell targeting. Until recently, H. pylori isolates were classified as tox+ or tox-, depending on whether they induced vacuoles in HeLa cells or not. Today, we know that almost all strains are cytotoxic. The major difference between tox+ and tox- resides in the cell binding domain, which exists in two allelic forms, only one of which is toxic for HeLa cells. The two forms, named m1 and m2, are found predominantly in Western and Chinese isolates respectively.

Bacterial Proteins↗
PubMed1999

Internalization and proteolytic action of botulinum toxins in CNS neurons and astrocytes.

Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red-conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins.

Animals↗
PubMed1999

Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis.

Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.

Amino Acid Sequence↗
PubMed1999

Functional characterisation of tetanus and botulinum neurotoxins binding domains.

Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial neurotoxins.

Animals↗
PubMed1999

Thrombotic thrombocytopenic purpura and autoimmunity: a tale of shadows and suspects.

BACKGROUND AND OBJECTIVE: The key pathogenic feature of TTP is the formation of platelet aggregates within the microcirculation; however, the etiology of such aggregates has been elusive for years. A large amount of evidence points to an abnormal interaction between damaged vascular endothelium and platelets, although the cause of the primary microvascular endothelial cell injury is seldom clear. The autoimmune hypothesis often recurs, and this is based on a number of observations: the claimed superiority of plasma-exchange over plasma infusion, the anecdotal report of the presence of immunocomplexes and autoantibodies in TTP patients, the efficacy of the administration of corticosteroids and other immunosuppressant agents, and the concomitant occurrence of TTP in association with autoimmune diseases, especially systemic lupus erythematosus (SLE). This review will focus on the complex relationships between TTP and humoral autoimmunity; in particular, similarities and differences between TTP, SLE and antiphospholipid (aPL) antibodies syndrome, as well as the putative role of several other antibodies directed towards endothelial cells and/or platelets, including the recently discovered anti-CD36 antibodies and antivWF-cleaving metalloprotease, will be discussed. DESIGN AND METHODS: The authors have been involved in the study and treatment of TTP and autoimmune diseases for years; furthermore, the PubMed data base of the National Library of Congress has been extensively searched using the Internet. CONCLUSIONS: Although over the years evidence has increased in favor of the autoimmune hypothesis for TTP etiopathogenesis, TTP should not yet be considered an autoimmune disease. Autoantibodies should be regarded as only one of the many different insults which can trigger microvascular thrombosis even though the autoimmune theory of the pathogenesis of TTP is gaining more and more strength. As far as concerns the relationship between TTP, SLE and aPL antibodies-related disorders, these diseases should be distinguished on the basis of both different clinical presentations and accurate antibody screening, although this approach should definitely not delay the prompt start of treatment.

ADAM Proteins↗
PubMed1998

In vitro biological activity and toxicity of tetanus and botulinum neurotoxins.

Tetanus and botulinum neurotoxins are the most potent toxins known and cause tetanus and botulism, respectively. They are zinc-endopeptidases acting in the cytosol, where they cleave SNARE proteins. Here, we report on the assay of their metalloproteolytic activity in vitro on recombinant SNARE proteins. We also describe the assay of their activity in nerve cells in culture using antibodies specific for the SNARE proteins. Together with recent reports from other laboratories, these results show that the toxicity of these powerful neurotoxins can be appropriately assayed in vitro, thus reducing considerably the number of animals currently used in the evaluation of the toxicity of tetanus toxoid vaccine and of the botulinum neurotoxins to be used for human therapy.

Animals↗
PubMed1998

Different time courses of recovery after poisoning with botulinum neurotoxin serotypes A and E in humans.

Botulinum toxin serotypes A and E (BoNT/A and /E) cleave the carboxy-terminus of synaptosomal associated protein-25 (SNAP-25) removing nine and 26 residues, respectively. To investigate the effect of these lesions of the same target molecule, 11 volunteers were injected with 3 IU of BoNT/A in the extensor digitorum brevis (EDB) muscle of one foot and with 3 IU of BoNT/E in the contralateral one. In addition, seven volunteers were similarly injected with mixtures of BoNT/A + BoNT/E. Compound muscular action potential (CMAP) was measured at different time intervals and the percentage variation of CMAP (%CMAP) was calculated. Unexpectedly, a much faster recovery of %CMAP after BoNT/E injections was observed. Double poisoned EBD muscles recovered similarly to BoNT/E. So, a larger deletion of the SNAP-25 molecule caused by BoNT/E leads to a faster functional recovery.

Action Potentials↗
PubMed1998

Cell vacuolization induced by Helicobacter pylori VacA toxin: cell line sensitivity and quantitative estimation.

A major virulence factor released by Helicobacter pylori is a protein toxin, termed VacA, which induces the formation of large intracellular vacuoles characterised by a lumenal acidic pH. Consequently they accumulate membrane permeable weak bases. The increase in neutral red uptake by intoxicated cells is the only known in vitro procedure to estimate quantitatively the activity of VacA. With the goal to standardize this assay, several parameters were evaluated: cell type, serum concentration, cell density and toxin concentration. Among the different cell types tested, HeLa cells were found to be the most sensitive to VacA. Results show that several factors contribute to VacA activity and that optimal vacuolation is achieved at non-confluent cell density, in the presence of low serum concentrations.

Bacterial Proteins↗
PubMed1998

TPA and butyrate increase cell sensitivity to the vacuolating toxin of Helicobacter pylori.

The Helicobacter pylori toxin VacA induces large membrane-bound vacuolar compartments of late endosomal/lysosomal origin. Pre-treatment of cells with TPA and butyrate enhances the toxin induced vacuolisation up to 20 times, depending on the cell line, whereas other differentiating factors such as DMSO, EGF, valeric and retinoic acid have no effect. The higher toxin sensitivity induced by TPA does not result from an increased surface binding or endocytosis. The effect of TPA is apparent after several hours from addition and is inhibited by a PKC specific inhibitor. These data suggest that expression of cellular proteins, other than the toxin receptor(s), influences the vacuolating activity of VacA and may contribute to the sensitivity of different cell lines. The present findings define the most sensitive in vitro assay of the activity of VacA.

Animals↗
PubMed1998

Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin.

The effects of the vacuolating toxin (VacA) released by pathogenic strains of Helicobacter pylori on several polarized epithelial monolayers were investigated. Trans-epithelial electric resistance (TER) of monolayers formed by canine kidney MDCK I, human gut T84, and murine mammary gland epH4, was lowered by acid-activated VacA. Independent of the cell type and of the starting TER value, VacA reduced it to a minimal value of 1,000-1,300 Omega x cm2. TER decrease was paralleled by a three- to fourfold increase of [14C]-mannitol (molecular weight 182.2) and a twofold increase of [14C]-sucrose (molecular weight 342.3) transmonolayer flux. On the contrary, transmembrane flux of the proinflammatory model tripeptide [14C]-N-formyl-Met-Leu-Phe (molecular weight 437.6), of [3H]-inuline (molecular weight 5,000) and of HRP (molecular weight 47,000) did not change. These data indicate that VacA increases paracellular epithelial permeability to molecules with molecular weight < 350-440. Accordingly, the epithelial permeability of Fe3+ and Ni2+ ions, essential for H. pylori survival in vivo, was also increased by VacA. High-resolution immunofluorescence and SDS-PAGE analysis failed to reveal alterations of junctional proteins ZO-1, occludin, cingulin, and E-cadherin. It is proposed that induction by VacA of a selective permeabilization of the epithelial paracellular route to low molecular weight molecules and ions may serve to supply nutrients, which favor H. pylori growth in vivo.

Animals↗
PubMed1998

Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages.

Lethal factor (LF) is the major virulence factor produced by Bacillus anthracis. LF is sufficient to cause death in laboratory animals and cytolysis of peritoneal macrophages and macrophage cell lines. LF contains the characteristic zinc binding motif of metalloproteases and indirect evidence suggest that this hydrolytic activity is essential for its cytotoxicity. To identify the substrate(s) of LF, we have used the yeast two-hybrid system, employing a LF inactive mutant as bait. This approach has led to the identification of the MAP kinase kinases (MAPKKs) Mek1 and Mek2 as proteins capable of specific interaction with LF. LF cleaves Mek1 and Mek2 within their N-terminus in vitro and in vivo, hydrolyzing a Pro8-Ile9 and a Pro10-Arg11 peptide bond in Mek1 and Mek2 respectively. The removal of the amino terminus of MAPKKs eliminates the "docking site" for the MAPKs ERK1 and ERK2, which become phosphorylated in cultured macrophages following toxin challenge. The possible implications of these findings for the cytolysis of macrophage cells induced by LF are discussed. These results open the way to the design and screening of specific inhibitors of LF.

Amino Acid Sequence↗
PubMed1998

The acid activation of Helicobacter pylori toxin VacA: structural and membrane binding studies.

The cell vacuolating activity of the protein toxin VacA, released by Helicobacter pylori, is strongly increased in vitro by exposure to acidic pH followed by neutralization. This short acid exposure does not increase significantly the binding of VacA to cell or to lipid membranes. However, membrane photolabeling with photoactivatable radioactive phospholipids and ANS binding studies show that VacA transiently exposed to pH equal or lower than 5 changes conformation and exposes on its surface hydrophobic segments. Both the 32 and the 58 kDa subunits of the toxin insert in the lipid bilayer and interact with the fatty acid chains of phospholipids. Membrane binding and penetration are enhanced by incubating target cells or liposomes with the toxin at mild acidic pH values, similar to those present around H. pylori on the stomach mucosa. These findings are discussed with respect to the critical step in cell intoxication consisting in the translocation of the active toxin domain into the cell cytosol. We suggest that membrane translocation takes place at the plasma membrane level.

Affinity Labels↗
PubMed1998

Selective inhibition of Ii-dependent antigen presentation by Helicobacter pylori toxin VacA.

A major virulence factor in the stomach chronic infection by Helicobacter pylori is a protein toxin (VacA), which alters cell membrane trafficking of late endosomal/prelysosomal compartments. Its role in the chronic infection established by H. pylori is unknown. To test the possibility that VacA alters antigen processing taking place in prelysosomal compartments, we have used the well-established model of antigen processing and presentation consisting of tetanus toxoid-specific human (CD4(+)) T cells stimulated by autologous antigen-pulsed Epstein-Barr virus-transformed B cells. We found that VacA interferes with proteolytic processing of tetanus toxin and toxoid and specifically inhibits the Ii-dependent pathway of antigen presentation mediated by newly synthesized major histocompatibility complex (MHC) class II, while leaving unaffected the presentation pathway dependent on recycling MHC class II. The results presented here suggest that VacA may contribute to the persistence of H. pylori by interfering with protective immunity and that this toxin is a new useful tool in the study of the different pathways of antigen presentation.

Antigen Presentation↗