Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain

ABSTRACT Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their...

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Autores principales:	Sabine Pellett, William H. Tepp, Marite Bradshaw, Suzanne R. Kalb, Janet K. Dykes, Guangyun Lin, Erin M. Nawrocki, Christina L. Pier, John R. Barr, Susan E. Maslanka, Eric A. Johnson
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2016
Materias:	BoNT/FA Clostridium botulinum botulinum neurotoxin chimeric toxin Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/4e275d261f0a4a04804f12f1bbdf5d14
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spelling	oai:doaj.org-article:4e275d261f0a4a04804f12f1bbdf5d142021-11-15T15:21:39ZPurification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain10.1128/mSphere.00100-152379-5042https://doaj.org/article/4e275d261f0a4a04804f12f1bbdf5d142016-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00100-15https://doaj.org/toc/2379-5042ABSTRACT Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.Sabine PellettWilliam H. TeppMarite BradshawSuzanne R. KalbJanet K. DykesGuangyun LinErin M. NawrockiChristina L. PierJohn R. BarrSusan E. MaslankaEric A. JohnsonAmerican Society for MicrobiologyarticleBoNT/FAClostridium botulinumbotulinum neurotoxinchimeric toxinMicrobiologyQR1-502ENmSphere, Vol 1, Iss 1 (2016)
institution	DOAJ
collection	DOAJ
language	EN
topic	BoNT/FA Clostridium botulinum botulinum neurotoxin chimeric toxin Microbiology QR1-502
spellingShingle	BoNT/FA Clostridium botulinum botulinum neurotoxin chimeric toxin Microbiology QR1-502 Sabine Pellett William H. Tepp Marite Bradshaw Suzanne R. Kalb Janet K. Dykes Guangyun Lin Erin M. Nawrocki Christina L. Pier John R. Barr Susan E. Maslanka Eric A. Johnson Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain
description	ABSTRACT Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.
format	article
author	Sabine Pellett William H. Tepp Marite Bradshaw Suzanne R. Kalb Janet K. Dykes Guangyun Lin Erin M. Nawrocki Christina L. Pier John R. Barr Susan E. Maslanka Eric A. Johnson
author_facet	Sabine Pellett William H. Tepp Marite Bradshaw Suzanne R. Kalb Janet K. Dykes Guangyun Lin Erin M. Nawrocki Christina L. Pier John R. Barr Susan E. Maslanka Eric A. Johnson
author_sort	Sabine Pellett
title	Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain
title_short	Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain
title_full	Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain
title_fullStr	Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain
title_full_unstemmed	Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain
title_sort	purification and characterization of botulinum neurotoxin fa from a genetically modified <named-content content-type="genus-species">clostridium botulinum</named-content> strain
publisher	American Society for Microbiology
publishDate	2016
url	https://doaj.org/article/4e275d261f0a4a04804f12f1bbdf5d14
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Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified <named-content content-type="genus-species">Clostridium botulinum</named-content> Strain

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