Purification of Clostridium botulinum Type A Neurotoxin

Malizio, Carl J.; Goodnough, Michael C.; Johnson, Eric A.

doi:10.1385/1-59259-052-7:27

Cited by 69 publications

(79 citation statements)

References 17 publications

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“…The 150-kDa holotoxins of BoNT/A1, -2, -3, and -5 were purified from C. botulinum strains Hall A hyper, Kyoto-F, CDC A3, and A661222 as previously described (23,(32)(33)(34). The purified toxins were stored in 40% glycerol-phosphate-buffered saline at Ϫ20°C until use.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, groups of four female ICR mice (Harlan) (18 to 20 g body weight) were injected intraperitoneally (i.p.) with 500 l of gelatin phosphate buffer (30 mM sodium phosphate [pH 6.3] and 0.2% gelatin) containing serial dilutions of the BoNT/A subtypes (32). Mice were observed for 4 days from the time of injection for botulism symptoms and death.…”

Section: Methodsmentioning

confidence: 99%

“…Surviving and dead mice were counted and specific activities calculated using the method of Reed and Muench (38). 3 U of BoNT/A1, -2, or -5 in 100 l of gelatin phosphate buffer (32), as has previously been described for BoNT/A3 (23). Three mice per subtype were injected and injections repeated as necessary.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

et al. 2013

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Botulinum neurotoxins (BoNTs) are synthesized by Clostridium botulinum and exist as seven immunologically distinct serotypes designated A through G. For most serotypes, several subtypes have now been described based on nominal differences in the amino acid sequences. BoNT/A1 is the most well-characterized subtype of the BoNT/A serotype, and many of its properties, including its potency, its prevalence as a food poison, and its utility as a pharmaceutical, have been thoroughly studied. In contrast, much remains unknown of the other BoNT/A subtypes. In this study, BoNT/A subtype 1 (BoNT/A1) to BoNT/A5 were characterized utilizing a mouse bioassay, an in vitro cleavage assay, and several neuronal cell-based assays. The data indicate that BoNT/A1 to -5 have distinct in vitro and in vivo toxicological properties and that, unlike those for BoNT/A1, the neuronal and mouse results for BoNT/A2 to -5 do not correlate with their enzymatic activity. These results indicate that BoNT/A1 to -5 have distinct characteristics, which are of importance for a greater understanding of botulism and for pharmaceutical applications.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

et al. 2013

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“…Pure botulinum neurotoxin type A1 was purified from C. botulinum strain Hall A hyper as described previously (28). The specific activity was determined via a mouse bioassay (17,40) to be 7.8 pg/mouse 50% lethal dose (LD 50 ).…”

Section: Methodsmentioning

confidence: 99%

Rapid Immune Responses to a Botulinum Neurotoxin Hc Subunit Vaccine through In Vivo Targeting to Antigen-Presenting Cells

et al. 2011

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“…Purified native BoNT/A and BoNT/E holotoxins were prepared from C. botulinum strains Hall A hyper (40) and Beluga E (41) as previously described and were purchased from Metabiologics or prepared in E.A.J. 's laboratory.…”

Section: Methodsmentioning

confidence: 99%

Bimodal modulation of the botulinum neurotoxin protein-conducting channel

Fischer

Nakai

Eubanks

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes.natural product ͉ protein translocation ͉ small molecule modulator

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Purification of Clostridium botulinum Type A Neurotoxin

Cited by 69 publications

References 17 publications

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

Rapid Immune Responses to a Botulinum Neurotoxin Hc Subunit Vaccine through In Vivo Targeting to Antigen-Presenting Cells

Bimodal modulation of the botulinum neurotoxin protein-conducting channel

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