Detection And Identification of Clostridium botulinum Neurotoxins

Hatheway, Charles L.; Ferreira, Joseph

doi:10.1007/978-1-4613-0361-9_39

Cited by 34 publications

(19 citation statements)

References 46 publications

(30 reference statements)

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“…The botulinum neurotoxin gene in this strain was inactivated using a ClosTron mutagenesis system (33, 34) as previously described (35). The resulting nontoxigenic C. botulinum strain Hall Ahyper/tox Ϫ was characterized by PCR and pulsed-field gel electrophoresis (PFGE) or Southern hybridizations to confirm insertion of the intron within the bont/A locus and by Western blotting and mouse bioassay (36) to confirm the absence of any toxin production.…”

Section: Methodsmentioning

confidence: 99%

“…A mouse bioassay was used to detect the presence of active botulinum neurotoxin in C. botulinum cultures and for determination of specific toxicity of purified rBoNT/A4 (36,40). For toxin detection in C. botulinum cultures, 96-h cultures were centrifuged at 12,000 ϫ g for 10 min to remove cellular debris, and 0.5-ml aliquots of culture supernatants were administered by intraperitoneal (i.p.)…”

Section: Methodsmentioning

confidence: 99%

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Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

Bradshaw

Tepp

Whitemarsh

et al. 2014

Appl Environ Microbiol

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Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100؋ lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

Bradshaw

Tepp

Whitemarsh

et al. 2014

Appl Environ Microbiol

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“…Strains were serotyped using an antibody capture enzyme-linked immunosorbent assay with serotype-specific monoclonal antibodies. In some cases, serotypes were confirmed using mouse neutralization (19). Silent (not expressed) BoNT/B genes were detected using real-time PCR (7).…”

Section: Strainsmentioning

confidence: 99%

Genetic Diversity among Botulinum Neurotoxin-Producing Clostridial Strains

et al. 2007

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Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.Clostridium botulinum is a taxonomic collection of several distinct species of anaerobic gram-positive spore-forming bacteria that produce the most poisonous substance known, botulinum neurotoxin (BoNT) (1,8). These organisms, along with related neurotoxin-producing species that, for a variety of reasons, were not included under the C. botulinum taxon, pose global health problems that affect both infant and adult humans and can also affect wildlife, waterfowl, and domestic animals. They cause intoxication through ingestion of the neurotoxin in contaminated foods. Toxicoinfections can also occur after contact with bacteria or bacterial spores (6, 17). These pathogens are ubiquitous and can be found in soils and sedi-

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“…2 The bont gene cluster can also be present in some C baratii strains (sometimes referred to as group V) and some C. butyricum strains (sometimes referred to as group VI). 3 The bont gene codes for a potent neurotoxin that inactivates anchoring of acetylcholine vesicles in neuromuscular junctions, thereby causing flaccid paralysis (botulism). Seven different toxinotypes (A-G) can be distinguished and some (B, E and F) are present in more than one of the BoNT-producing Clostridium groups.…”

Section: Horizontal Gene Transfer Of Toxin Genes In Clostridium Botulmentioning

confidence: 99%