Botolinum toxins: their structure, properties, and genetics (original) (raw)
Related papers
Botulinum neurotoxins: genetic, structural and mechanistic insights
Nature Reviews Microbiology, 2014
Clostridium is a genus of sporulating and anaerobic Gram-positive, rod-shaped bacteria that includes more than 150 species. These bacteria are widely distributed in the environment and in anaerobic regions of the intestines of several animals, where they are typically found as spores, which are resistant to physical and chemical stresses and can persist for long periods of time until favourable conditions enable germination 1,2 . Under appropriate environmental conditions (such as humidity, nutrients and the absence of oxygen), spores germinate into vegetative cells; conversely, exposure to oxygen, as well as water and nutrient deprivation, trigger sporulation. Several clostridia, including Clostridium difficile, Clostridium perfringens and Clostridium sordelli, are pathogenic, owing to the release of protein toxins, but only a few species are neurotoxigenic. For example, Clostridium tetani produces tetanus neurotoxin, which blocks neurotransmitter release in spinal cord interneurons and causes the spastic paralysis of tetanus 3 . In addition, six phylogenetically distinct clostridia produce more than 40 different botulinum neurotoxins (BoNTs) (BOX 1). BoNTs consist of three primary domains: two of these domains enable binding to nerve terminals and translocation of the toxin into the neuronal cytosol, and the third domain comprises a metalloprotease that inhibits the release of neurotransmitter by peripheral nerve terminals (BOX 2), which causes the flaccid paralysis and autonomic dysfunctions that are typical of botulism 2,4 . The neurospecificity and toxic potency of BoNTs make them the most powerful known toxins, and they are potential bioterrorism weapons 5,6 . By contrast, their absolute neurospecificity has enabled BoNTs to be used as effective therapeutic agents for human diseases that Neurotransmitter An endogenous chemical that transmits signals across a synapse from a neuron to a postsynaptic cell.
The Botulinum Neurotoxin Complex and the Role of Ancillary Proteins
Molecular Aspects of Botulinum Neurotoxin, 2014
All seven known serotypes of botulinum neurotoxin (BoNT) are produced in the form of a complex with a group of neurotoxin-associated proteins (NAPs). The BoNT complex is encoded by a gene cluster regulated by its own transcription factor, and the proteins coded by polycistronic messenger ribonucleic acid (mRNA) self-assemble into complexes of 300-900 kDa. Types A, B, C, D, and G complexes contain hemagglutinin (HA), whereas types E and F complexes do not contain HA. Sequence homology among respective BoNTs and NAPs range from 55.3 to 98.5 %, and all the proteins in the BoNT complex belong to a stable class of protein with high longevity inside mammalian cells. A new 250-kDa protein (P-250) with high immunogenicity has been identified in the BoNT/A complex which is not part of the neurotoxin gene cluster. The 33-kDa hemagglutinin (HA-33) is the most abundant NAP. The HA-33 is protease resistant and is highly immunogenic. HA-33 appears to play an important role in the translocation of the neurotoxin across the gut wall, enhancing the endopeptidase activity of BoNT and protection of BoNT against proteases. The role of other NAPs is not as clear, and their role in the biology of the bacteria is not understood at all. BoNT complexes are used as therapeutic products, although a therapeutic product without NAPs appears to retain the properties of the complex-based products. NAPs in therapeutic products may have other subtle long-term effects which need to be investigated. Keywords Botulinum • Botox • Clostridium • Dysport • Complex • Neurotoxin • NAPs • Protein stability • Serotypes • Therapeutic • Toxin • Xeomin • Botulinum neurotoxin • Neurotoxin-associated proteins • Hemagglutinin • Progenitor neurotoxin • Gene cluster • Operon • Polycistronic • Molecular stoichiometry • Endopeptidase K. A. Foster (ed.), Molecular Aspects of Botulinum Neurotoxin, Current Topics in Neurotoxicity 4,
A historical and proteomic analysis of botulinum neurotoxin type/G
BMC Microbiology, 2011
Background: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. Results: An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs-nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.
Molecular assembly of botulinum neurotoxin progenitor complexes
Proceedings of the National Academy of Sciences, 2013
Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum and associates with nontoxic neurotoxin-associated proteins to form high-molecular weight progenitor complexes (PCs). The PCs are required for the oral toxicity of BoNT in the context of food-borne botulism and are thought to protect BoNT from destruction in the gastrointestinal tract and aid in absorption from the gut lumen. The PC can differ in size and protein content depending on the C. botulinum strain. The oral toxicity of the BoNT PC increases as the size of the PC increases, but the molecular architecture of these large complexes and how they contribute to BoNT toxicity have not been elucidated. We have generated 2D images of PCs from strains producing BoNT serotypes A1, B, and E using negative stain electron microscopy and single-particle averaging. The BoNT/A1 and BoNT/B PCs were observed as ovoid-shaped bodies with three appendages, whereas the BoNT/E PC was observed as an ovoid body. Both the BoNT/A1 and Bo...
Separation of the components of type A botulinum neurotoxin complex by electrophoresis
Toxicon, 2003
Clostridium botulinum neurotoxins (BoNTs) are the most toxic substances known. They exert potent neuroparalysis on vertebrates. C. botulinum produces seven serotypes of neurotoxin (A-G). BoNT/A, found in bacterial cultures of C. botulinum type A, is produced as a complex with a group of neurotoxin associated proteins (NAPs). Botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against the acidity and proteases of the stomach. Here, we used sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS -PAGE) for separation and identification of the constituent proteins of BoNT/A complex. A range of homogenous and gradient SDS -PAGE gels was used to resolve the BoNT/A complex. These gels were run under constant voltage and constant current conditions. The molecular weight and relative amount of each protein band were determined. On a 12.5% homogenous SDS -PAGE under reducing conditions, seven protein bands were identified with average molecular weights of 118, 106, 90, 56, 36, 23 and 17 kDa. The relative amounts of these seven proteins were determined densitometrically as 10, 6, 13, 27, 22, 13 and 8%, respectively. The separation and identification of BoNT/A complex will help in understanding the molecular structure and function of BoNT/A NAPs and their interaction with the toxin, in the toxico-infection process of the botulism diseased state. In particular, the stoichiometry of the individual components is established for a typical preparation of BoNT/A complex. Furthermore, the studies reported here identify the most favorable conditions for the baseline resolution of BoNT/A NAPs proteins for other workers in this field. q
2010
Botulinum neurotoxins (BoNTs) are highly potent poisons produced by seven serotypes of Clostridium botulinum. The mechanism of neurotoxin action is a multistep process which leads to the cleavage of one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contain a coiled-coil domain while types B and type G neurotoxin do not. Interestingly, phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G are closely related. These results suggest that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomerisation of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication.
Applied Sciences
Botulinum toxins or neurotoxins (BoNTs) are the most potent neurotoxins known, and are currently extensively studied, not only for their potential lethality, but also for their possible therapeutic and cosmetic uses. Currently, seven types of antigenically distinct toxins are known and characterized, produced by a rod-shaped bacterium, Clostridium botulinum. Human poisoning by botulism (presenting with severe neuromuscular paralytic disease) is usually caused by toxins A, B, E, and F type. Poisoning from contaminated food preparations is the most common cause of noniatrogenic botulism. The spores are highly resistant to heat but are easily destroyed at 80 °C for thirty minutes. Type A and B toxins are resistant to digestion by the enzymes of the gastrointestinal system. After their entry, BoNTs irreversibly bind to cholinergic nerve endings and block the release of acetylcholine from the synapses. In contrast, in wound botulism, the neurotoxin is instead product by the growth of C.b...
Toxins
Botulinum neurotoxins (BoNTs) cause flaccid neuromuscular paralysis by cleaving one of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. BoNTs display high affinity and specificity for neuromuscular junctions, making them one of the most potent neurotoxins known to date. There are seven serologically distinct BoNTs (serotypes BoNT/A to BoNT/G) which can be further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) based on small changes in their amino acid sequence. Of these, BoNT/A1 and BoNT/B1 have been utilised to treat various diseases associated with spasticity and hypersecretion. There are potentially many more BoNT variants with differing toxicological profiles that may display other therapeutic benefits. This review is focused on the structural analysis of the cell-binding domain from BoNT/A1 to BoNT/A6 subtypes (HC/A1 to HC/A6), including features such as a ganglioside binding site (GBS), a dynamic loop, a synaptic vesicle glyc...