Cesare Montecucco

Mechanism of action of tetanus and botulinum neurotoxins

The clostridial neurotoxins responsible for tetanus and botulism are metallo‐proteases that enter nerve cells and block neurotransmitter release via zinc‐dependent cleavage of protein components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular Junction and is internalized and transported retroaxonally to the spinal cord. Whilst TeNT causes spastic paralysis by acting on the spinal inhibitory interneurons, the seven serotypes of botullnum neurotoxins (BoNT) induce a flaccid paralysis because they intoxicate the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G specifically cleave VAMP/synaptobrevin, a membrane protein of small synaptic vesicles, at different single peptide bonds. Proteins of the presynaptic membrane are specifically attacked by the other BoNTs: serotypes A and E cleave SNAP‐25 at two different sites located within the carboxyl terminus, whereas the specific target of serotype C is syntaxin.

Structure and function of tetanus and botulinum neurotoxins

Tetanus and botulinum neurotoxins are produced by Clostridia and cause the neuroparalytic syndromes of tetanus and botulism. Tetanus neurotoxin acts mainly at the CNS synapse, while the seven botulinum neurotoxins act peripherally. Clostridial neurotoxins share a similar mechanism of cell intoxication: they block the release of neurotransmitters. They are composed of two disulfide-linked polypeptide chains. The larger subunit is responsible for neurospecific binding and cell penetration. Reduction releases the smaller chain in the neuronal cytosol, where it displays its zinc-endopeptidase activity specific for protein components of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxins B, D, F and G recognize specifically VAMP/ synaptobrevin. This integral protein of the synaptic vesicle membrane is cleaved at single peptide bonds, which differ for each neurotoxin. Botulinum A, and E neurotoxins recognize and cleave specifically SNAP-25, a protein of the presynaptic membrane, at two different sites within the carboxyl-terminus. Botulinum neurotoxin type C cleaves syntaxin, another protein of the nerve plasmalemma. These results indicate that VAMP, SNAP-25 and syntaxin play a central role in neuroexocytosis. These three proteins are conserved from yeast to humans and are essential in a variety of docking and fusion events in every cell. Tetanus and botulinum neurotoxins form a new group of zinc-endopeptidases with characteristic sequence, mode of zinc coordination, mechanism of activation and target recognition. They will be of great value in the unravelling of the mechanisms of exocytosis and endocytosis, as they are in the clinical treatment of dystonias.

Living dangerously: how Helicobacter pylori survives in the human stomach

Helicobacter pylori was already present in the stomach of primitive humans as they left Africa and spread through the world. Today, it still chronically infects more than 50% of the human population, causing, in some cases, severe diseases such as peptic ulcers and stomach cancer. To succeed in these long-term associations, H. pylori has developed a unique set of virulence factors, which allow survival in a unique and hostile ecological niche — the human stomach.

Botulinum neurotoxins serotypes A and E cleave SNAP‐25 at distinct COOH‐terminal peptide bonds

SNAP‐25, a membrane‐associated protein of the nerve terminal, is specifically cleaved by botulinum neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo‐endopeptidase toxins cleave SNAP‐25 at two distinct carboxyl‐terminal sites. Serotype A catalyses the hydrolysis of the Gln197‐Arg198 peptide bond, while serotype E cleaves the Arg180‐Ile181 peptide linkage. These results indicate that the carboxyl‐terminal region of SNAP‐25 plays a crucial role in the multi‐protein complex that mediates vesicle docking and fusion at the nerve terminal.

Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNγ-induced release of NO and TNFα

The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in animals and cause macrophage lysis. LF is a zinc‐endopeptidase which cleaves two mitogen‐activated proten kinase kinases (MAPKKs), Mek1 and Mek2, within the cytosol. Here, we show that also MKK3, another dual‐specificity kinase that phosphorylates and activates p38 MAP kinase, is cleaved by LF in macrophages. No direct correlation between LF‐induced cell death and cleavage of these MAPKKs was found in macrophage cell lines and primary peritoneal cells exhibiting different sensitivity to LF. However, we present the first evidence that sublytic doses of LF cleave Meks and cause a substantial reduction in the production of NO and tumour necrosis factor‐α induced by lipopolysaccharide/interferonγ. We suggest that this effect of LF is relevant during the first stages of B. anthracis infection, when a reduction of the inflammatory response would permit growth and diffusion of the bacterium.

The use of acetylated ferricytochrome C for the detection of superoxide radicals produced in biological membranes

Summary Acetylation of 60% of lysine residues of horse heart ferricytochrome c results in more than 95% decrease of its ability to be reduced by mitochondrial and microsomal reductases and to become oxidized (after chemical reduction) by mitochondrial oxidase. The ability of acetylated ferricytochrome c to be reduced by 0 2 − radicals is maintained, making this derivative useful for the detection of 0 2 − radicals in biological systems containing cytochrome c reductases or oxidases. Mitochondrial membranes can reduce acetylated ferricytochrome c at a rate of 0.5 nmoles.min −1 .mg −1 . Such a reaction is 82% inhibited by 2.8 × 10 −8 M superoxide dismutase.

Formation of anion‐selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity

The vacuolating toxin VacA, a major determinant of Helicobacter pylori‐associated gastric diseases, forms anion‐selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low‐conductance VacA pores in the cell plasma membrane and not to the opening of Ca2+‐ or volume‐activated chloride channels. VacA‐dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB), while2‐[(2‐cyclopentenyl‐6,7dichloro‐2,3‐dihydro‐2‐methyl‐1‐oxo‐1H‐inden‐5‐yl)oxy]acetic acid (IAA‐94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA‐94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans‐epithelial conductivity.

Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells

We used an in vitro system based on streptolysin O-permeabilized MDCK cells to study the involvement of NSF, SNAP, SNAREs, and Rab proteins in polarized membrane transport of epithelial cells. In MDCK cells, transport from the trans-Golgi network (TGN) to the basolateral plasma membrane is inhibited by anti-NSF antibodies and stimulated by alpha-SNAP. In contrast, transport from the TGN to the apical cell surface is not affected by anti-NSF antibodies or alpha-SNAP. Furthermore, apical transport is insensitive to Rab-GDI and tetanus and botulinum neurotoxins, which inhibit basolateral transport. These results provide evidence that the Rab-NSF-SNAP-SNARE mechanism operates in basolateral transport, while other molecules constitute the machinery for vesicular delivery in the apical pathway.

Botulinum neurotoxins: genetic, structural and mechanistic insights

Botulinum neurotoxins (BoNTs) are produced by anaerobic bacteria of the genus Clostridium and cause a persistent paralysis of peripheral nerve terminals, which is known as botulism. Neurotoxigenic clostridia belong to six phylogenetically distinct groups and produce more than 40 different BoNT types, which inactivate neurotransmitter release owing to their metalloprotease activity. In this Review, we discuss recent studies that have improved our understanding of the genetics and structure of BoNT complexes. We also describe recent insights into the mechanisms of BoNT entry into the general circulation, neuronal binding, membrane translocation and neuroparalysis.

The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin

The VacA cytotoxin, produced by toxigenic strains of Helicobacter pylori, induces the formation of large vacuoles highly enriched in the small GTPase rab7. To probe the role of rab7 in vacuolization, HeLa cells were transfected with a series of rab mutants and exposed to VacA. Dominant‐negative mutants of rab7 effectively prevented vacuolization, whereas homologous rab5 and rab9 mutants were only partially inhibitory or ineffective, respectively. Expression of wild‐type or GTPase‐deficient rab mutants synergized with VacA in inducing vacuolization. In vitro fusion of late endosomes was enhanced by active rab7 and inhibited by inactive rab7, consistent with vacuole formation by merging of late endosomes in a process that requires functional rab7. Taken together, the effects of overexpressed rab proteins described here indicate that continuous membrane flow along the endocytic pathway is necessary for vacuole growth.