Cell membrane Complex Enzyme Eukaryotic cell or cell component Microbe-host cell complex Microorganism or its component Other Other -- ion Pathway or action Protein Protein or gene Protein or gene complex Protein or protein complex Bacterial membrane or virus envelope Cell membrane Cytoplasm Eukaryotic cell or cell component Extracellular Golgi Golgi membrane Intercellular Intracellular Mitochondria Nucleocapsid/Cytoplasm Organelle Organelle -- Cell membrane Organelle -- Endoplasmic reticulum Organelle -- ER Organelle -- Golgi Organelle -- Golgi membrane Organelle -- Nucleus Organelle -- Phagolysosome Organelle -- Phagosome Organelle -- Ribosome Other Phagolysosome Phagosome Chaperone Defense, immunity protein Enzyme Enzyme activator Enzyme inhibitor Genomic S segment Infection Ligand binding or carrier Motor Nucleic acid binding Other Signal transducer Toxicity Transcription factor binding Transcription regulation Transporter Unknown IC Inferred by Curator IDA Inferred from Direct Assay IEA Inferred from Electronic Annotation IEP Inferred from Expression Pattern IGI Inferred from Genetic Interaction IMP Inferred from Mutant Phenotype IPI Inferred from Physical Interaction ISS Inferred from Sequence or Structural Similarity NAS Non-traceable Author Statement ND No biological Data available RCA inferred from Reviewed Computational Analysis TAS Traceable Author Statement NR Not Recorded Matarasso SL Dermatologic surgery : official publication for American Society for Dermatologic Surgery [et al.] Comparison of botulinum toxin types A and B: a bilateral and double-blind randomized evaluation in the treatment of canthal rhytides 2003 12534505 PubMed Maksymowych AB, Simpson LL The Journal of pharmacology and experimental therapeutics Structural features of the botulinum neurotoxin molecule that govern binding and transcytosis across polarized human intestinal epithelial cells 2004 15140915 PubMed Kalandakanond S, Coffield JA The Journal of pharmacology and experimental therapeutics Cleavage of SNAP-25 by botulinum toxin type A requires receptor-mediated endocytosis, pH-dependent translocation, and zinc 2001 11181932 PubMed Maksymowych AB, Simpson LL The Journal of biological chemistry Binding and transcytosis of botulinum neurotoxin by polarized human colon carcinoma cells 199821 9705335 PubMed Maksymowych AB, Reinhard M, Malizio CJ, Goodnough MC, Johnson EA, Simpson LL Infection and immunity Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade 1999 10456920 PubMed Anne C, Turcaud S, Quancard J, Teffo F, Meudal H, Fournie-Zaluski MC, Roques BP Journal of medicinal chemistry Development of potent inhibitors of botulinum neurotoxin type B 200323 14561084 PubMed Segelke B, Knapp M, Kadkhodayan S, Balhorn R, Rupp B Proceedings of the National Academy of Sciences of the United States of America Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state: Evidence for noncanonical zinc protease activity 20044 15107500 PubMed Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Hauer J, Layton M, Lillibridge S, Osterholm MT, O'Toole T, Parker G, Perl TM, Russell PK, Swerdlow DL, Tonat K JAMA : the journal of the American Medical Association Botulinum toxin as a biological weapon: medical and public health management 200128 11209178 PubMed Brin MF Muscle & nerve. Supplement Botulinum toxin: chemistry, pharmacology, toxicity, and immunology 1997 9826987 PubMed Pellizzari R, Rossetto O, Schiavo G, Montecucco C Philosophical transactions of the Royal Society of London. Series B, Biological sciences Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses 199928 10212474 PubMed Kouguchi H, Watanabe T, Sagane Y, Sunagawa H, Ohyama T The Journal of biological chemistry In vitro reconstitution of the Clostridium botulinum type D progenitor toxin 200225 11713244 PubMed Park JB, Simpson LL Infection and immunity Inhalational poisoning by botulinum toxin and inhalation vaccination with its heavy-chain component 2003 12595426 PubMed Simpson LL Annual review of pharmacology and toxicology Identification of the major steps in botulinum toxin action 2004 14744243 PubMed Clostridium botulinum Function: Toxicity. Botulinum toxin is one of the most potent neurotoxins known, which is synthesized by the bacteria Clostridium botulinum. The great majority of cases of botulism are contracted orally, either by ingesting food containing botulinum toxin or by ingesting food contaminated with Clostridium botulinum bacteria or spores that reproduce in the gut and release the toxin. The various etiologies of botulism can be divided into two broad categories: primary intoxication and primary infection followed by secondary intoxication.(Simpson, 2004) Botulinum protoxin Function: Enzyme. Botulinum protoxin is synthesized as a single-chain polypeptide with a molecular mass of 150 kDa.(Park et al., 2003) Protoxin. Nicking by protease Function: Enzyme. This relatively inactive protoxin undergoes posttranslation modification, during which a trypsin-like enzyme cleaves (nicks) the single-chain molecule to generate a dichain molecule in which a heavy-chain polypeptide (100 kDa, HC-100) is linked by a disulfide bond to a light-chain polypeptide (50 kDa, LC-50). The dichain molecule is the agent responsible for the disease botulism.(Park et al., 2003) Progenitor toxin complex. Formation and release Function: Enzyme. Neurotoxin is synthesized in seven immunologically distinct forms designated A, B, C, D, E, F, and G. Under natural conditions, each of these serotypes is released from clostridia in association with two classes of proteins: a family of hemagglutinins (HA) and a single nontoxin, nonhemagglutinin (NTNH). The complex that is composed of a toxin and noncovalently associated proteins is known as progenitor toxin. These nontoxic proteins may enhance toxicity, compared to toxin without the proteins, possibly by protecting the neurotoxin from proteolytic enzymes in the gut. Each of the serotypes responsible for human illness (mainly A, B, and E) forms a unique progenitor complex with associated proteins, but these associated proteins play no role in the signs and symptoms of clinical botulism.(Park et al., 2003)(Kouguchi et al., 2002)(Brin, 1997) Botulinum toxin. Food infection/Primary intoxication Function: Enzyme. The contaminated food serves as a culture medium for bacteria. The microorganisms mature and undergo autolysis and then release toxin. In primary intoxication, the patient ingests a food that is already tainted with botulinum toxin.(Simpson, 2004) Botulinum toxin. Primary human infection/Secondary intoxication Function: Enzyme. In primary infection, the patient ingests food that harbors botulinum spores that may germinate and colonize in the gut. In the process of growth, division, and autolysis, microorganisms release toxin in situ, and this leads to secondary intoxication. Consumption of botulinum spores is probably somewhat common, although the incidence of secondary intoxication is low. This can be attributed to several factors, with the most important being the inability of C. botulinum to compete for a niche in healthy guts that have a normal flora. This explains the fact that primary infection with secondary intoxication occurs almost exclusively in young infants (e.g., the normal flora has not developed) or in older persons who have been treated with certain antibiotics (e.g., the normal flora has been reduced or eliminated).(Simpson, 2004) Transport cells. Toxin binding to intestinal epithelial cell surface Function: Enzyme. The two broad categories of etiology, primary or secondary intoxication, have one key feature in common: toxin in the lumen of the gut must penetrate membrane barriers to reach blood and lymph. Toxin molecules, certainly progenitor complexes of toxin or HA and NTNH, are too large for any significant rate of transcellular diffusion. It is likely that the toxin binds to a cell surface receptor that is linked to an efficient transport process.At initial stage botulinum toxin interacts with epithelial cells (namely, transport cells). The two logical choices of transport cells are absorptive epithelial cells and M cells. Both cell types, along with intercellular tight junctions, serve the purpose of creating a barrier between the lumen of the gut and the general circulation. Both cell types have the ability to capture certain molecules or macromolecular complexes and transport them.(Simpson, 2004) Absorptive cells Function: Transporter. In the case of absorptive cells, the transport process typically does nothing more than ferry molecules across structural barriers. However, the major route utilized by toxin is transcytosis across absorptive cells. Toxin can bind to the apical surface of cells, undergo transcytosis, and be released on the basolateral side. Toxin that undergoes transport is released in a form that is structurally and functionally indistinguishable from that originally added to cells.(Simpson, 2004) M cells Function: Transporter. In the case of M cells, the transport process can be more involved. The M cell Peyer's Patch complex can capture and process molecules, including partial metabolic breakdown, to present potential antigens to the mucosal immune system. M cells could outweigh the contribution of absorptive cells only if their transport rates were vastly higher. Thus, absorptive cells are the major participants in botulinum toxin transport.(Simpson, 2004) Receptor-Toxin. Toxin and target cell binding Function: Transporter. From the site of transport cells, botulinum toxin diffuses in the body fluids, up to the presynaptic membrane of cholinergic endings where it binds.The receptor for botulinum toxin at the neuromuscular junction has not been unequivocally identified. Sialic acid-containing molecules, and perhaps gangliosides, might be implicated in toxin binding.It is clear now, that the botulinum toxin binds with high affinity to membrane; the binding domain of the toxin is localized within the heavy chain; and with the exception of serotypes C and D, which are closely related, have their own binding sites that are partially or wholly unique.(Simpson, 2004) Nerve terminus cytosol. Toxin penetration to the nerve terminus cytosol Function: Transporter. Since the light chains of botulinum toxins block neuroexocytosis by acting in the cytosol, at least this part of the toxin must reach the cell cytosol. All available evidence indicates that botulinum toxins do not enter the cell directly from the plasma membrane. Rather, they are endocytosed inside acidic cellular compartments.(Pellizzari et al., 1999) Plasma membrane. Toxin penetration by receptor-mediated endocytosis Function: Transporter. Botulinum toxin binds irreversibly to the presynaptic nerve ending - the heavy chain facilitates the binding and internalization of the toxin molecules into cholinergic neurons.Botulinum toxin enters the cells by receptor-mediated endocytosis and shortly thereafter escapes endosomes to act locally in the cytosol. For botulinum toxin, escape is known to be a pH-dependent phenomenon.(Simpson, 2004) Endosome membrane. Toxin penetration by pH-induced translocation Function: Transporter. The botulinum toxin membrane translocation process includes, probably, some distinct events. These include a pH-induced change in toxin structure that results in exposure of previously occult hydrophobic domains; insertion of the toxin into the endosome membrane; translocation of the light chain from the lumenal to the cytosolic surface of the membrane; reduction of the single disulfide bond that links the heavy chain and light chain; uncoupling of the noncovalent forces that bond the heavy and light chains, with subsequent separation of chains; and restoration of light-chain structure associated with movement from an acidic environment (endosome) to a more neutral environment (cytosol).(Simpson, 2004) Toxin (Neurotoxin). Expression of toxicity-blocking acetylcholine release Function: Enzyme. At normal neuromuscular transmission the SNARE (Soluble NSF-attachment Protein Receptor) proteins (SNAP-25 (Synaptosomal-Associated Protein of 25 kd), synaptobrevin (Vesicle-Associated Membrane Protein, or VAMP) and syntaxin) form a synaptic fusion complex which enables acetylcholine to be released into the neuromuscular junction. Release of acetylcholine at the neuromuscular junction is mediated by the assembly of a synaptic fusion complex that allows the membrane of the synaptic vesicle containing acetylcholine to fuse with the neuronal cell membrane. After membrane fusion, acetylcholine is released into the synaptic cleft and then bound by receptors on the muscle cell. The light chains of botulinum toxins are unique zinc proteases that have stringent substrate specificity and require exceptionally long substrates. From 15107500The light chains cleave specific sites on the SNARE proteins, preventing complete assembly of the synaptic fusion complex and thereby blocking acetylcholine release. Botulinum toxins types B, D, F, and G cleave synaptobrevin; types A, C, and E cleave SNAP-25; and type C cleaves syntaxin. Without acetylcholine release, the muscle is unable to contract and move.(Arnon et al., 2001)(Segelke et al., 2004) Neuromuscular junctions. Expression of toxicity-flaccid paralysis Function: . The neurotransmitter acetylcholine is not released into neuromuscular junctions, leading to paralysis of myosin filaments. Blockade of transmitter release accounts for flaccid paralysis and autonomic dysfunction that is characteristic of the botulism disease. In untreated persons, paralysis extends beyond; death results from airway obstruction (pharyngeal and upper airway muscle paralysis) and inadequate tidal volume (diaphragmatic and accessory respiratory muscle paralysis). Although the toxin acts preferentially on cholinergic nerve endings, it does have the ability to block exocytosis from other nerve endings as well.(Anne et al., 2003)