Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6

ABSTRACT Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals...

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Autores principales:	Molly S. Moritz, William H. Tepp, Marite Bradshaw, Eric A. Johnson, Sabine Pellett
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	BoNT BoNT/A6 botulinum neurotoxin cell entry duration potency Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a7f41dbd5a6b4b2081c2e2322e9b7527
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spelling	oai:doaj.org-article:a7f41dbd5a6b4b2081c2e2322e9b75272021-11-15T15:22:26ZIsolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 610.1128/mSphere.00466-182379-5042https://doaj.org/article/a7f41dbd5a6b4b2081c2e2322e9b75272018-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00466-18https://doaj.org/toc/2379-5042ABSTRACT Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals used to treat more than 200 neuronal disorders. BoNTs comprise 7 serotypes and more than 40 isoforms (subtypes). BoNT/A1 is the only A-subtype used clinically due to its high potency and long duration of action. While other BoNT/A subtypes have been purified and described, only BoNT/A2 is being investigated as an alternative to BoNT/A1. Here we describe subtype BoNT/A6 with improved pharmacological properties compared to BoNT/A1. It was isolated from Clostridium botulinum CDC41370, which produces both BoNT/B2 and BoNT/A6. The gene encoding BoNT/B2 was genetically inactivated, and A6 was isolated to greater than 95% purity. A6 was highly potent in cultured primary rodent neuronal cultures and in human induced pluripotent stem cell-derived neurons, requiring 20-fold less toxin to cause 50% SNAP-25 cleavage than A1. Second, A6 entered hiPSCs faster and more efficiently than A1 and yet had a long duration of action similar to BoNT/A1. Third, BoNT/A6 had similar LD50 as BoNT/A1 after intraperitoneal injection in mice; however, local intramuscular injection resulted in less systemic toxicity than BoNT/A1 and a higher (i.m.) LD50, indicating its potential as a safer pharmaceutical. These data suggest novel characteristics of BoNT/A6 and its potential as an improved pharmaceutical due to more efficient neuronal cell entry, greater ability to remain localized at the injection site, and a long duration. IMPORTANCE Botulinum neurotoxins (BoNTs) have proved to be an effective treatment for a large number of neuropathic conditions. BoNTs comprise a large family of zinc metalloproteases, but BoNT/A1 is used nearly exclusively for pharmaceutical purposes. The genetic inactivation of a second BoNT gene in the native strain enabled expression and isolation of a single BoNT/A6 from cultures. Its characterization indicated that BoNT/A subtype A6 has a long duration of action comparable to A1, while it enters neurons faster and more efficiently and remains more localized after intramuscular injection. These characteristics of BoNT/A6 are of interest for potential use of BoNT/A6 as a novel BoNT-based therapeutic that is effective and has a fast onset, an improved safety profile, and a long duration of action. Use of BoNT/A6 as a pharmaceutical also has the potential to reveal novel treatment motifs compared to currently used treatments.Molly S. MoritzWilliam H. TeppMarite BradshawEric A. JohnsonSabine PellettAmerican Society for MicrobiologyarticleBoNTBoNT/A6botulinum neurotoxincell entrydurationpotencyMicrobiologyQR1-502ENmSphere, Vol 3, Iss 5 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	BoNT BoNT/A6 botulinum neurotoxin cell entry duration potency Microbiology QR1-502
spellingShingle	BoNT BoNT/A6 botulinum neurotoxin cell entry duration potency Microbiology QR1-502 Molly S. Moritz William H. Tepp Marite Bradshaw Eric A. Johnson Sabine Pellett Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6
description	ABSTRACT Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals used to treat more than 200 neuronal disorders. BoNTs comprise 7 serotypes and more than 40 isoforms (subtypes). BoNT/A1 is the only A-subtype used clinically due to its high potency and long duration of action. While other BoNT/A subtypes have been purified and described, only BoNT/A2 is being investigated as an alternative to BoNT/A1. Here we describe subtype BoNT/A6 with improved pharmacological properties compared to BoNT/A1. It was isolated from Clostridium botulinum CDC41370, which produces both BoNT/B2 and BoNT/A6. The gene encoding BoNT/B2 was genetically inactivated, and A6 was isolated to greater than 95% purity. A6 was highly potent in cultured primary rodent neuronal cultures and in human induced pluripotent stem cell-derived neurons, requiring 20-fold less toxin to cause 50% SNAP-25 cleavage than A1. Second, A6 entered hiPSCs faster and more efficiently than A1 and yet had a long duration of action similar to BoNT/A1. Third, BoNT/A6 had similar LD50 as BoNT/A1 after intraperitoneal injection in mice; however, local intramuscular injection resulted in less systemic toxicity than BoNT/A1 and a higher (i.m.) LD50, indicating its potential as a safer pharmaceutical. These data suggest novel characteristics of BoNT/A6 and its potential as an improved pharmaceutical due to more efficient neuronal cell entry, greater ability to remain localized at the injection site, and a long duration. IMPORTANCE Botulinum neurotoxins (BoNTs) have proved to be an effective treatment for a large number of neuropathic conditions. BoNTs comprise a large family of zinc metalloproteases, but BoNT/A1 is used nearly exclusively for pharmaceutical purposes. The genetic inactivation of a second BoNT gene in the native strain enabled expression and isolation of a single BoNT/A6 from cultures. Its characterization indicated that BoNT/A subtype A6 has a long duration of action comparable to A1, while it enters neurons faster and more efficiently and remains more localized after intramuscular injection. These characteristics of BoNT/A6 are of interest for potential use of BoNT/A6 as a novel BoNT-based therapeutic that is effective and has a fast onset, an improved safety profile, and a long duration of action. Use of BoNT/A6 as a pharmaceutical also has the potential to reveal novel treatment motifs compared to currently used treatments.
format	article
author	Molly S. Moritz William H. Tepp Marite Bradshaw Eric A. Johnson Sabine Pellett
author_facet	Molly S. Moritz William H. Tepp Marite Bradshaw Eric A. Johnson Sabine Pellett
author_sort	Molly S. Moritz
title	Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6
title_short	Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6
title_full	Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6
title_fullStr	Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6
title_full_unstemmed	Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6
title_sort	isolation and characterization of the novel botulinum neurotoxin a subtype 6
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/a7f41dbd5a6b4b2081c2e2322e9b7527
work_keys_str_mv	AT mollysmoritz isolationandcharacterizationofthenovelbotulinumneurotoxinasubtype6 AT williamhtepp isolationandcharacterizationofthenovelbotulinumneurotoxinasubtype6 AT maritebradshaw isolationandcharacterizationofthenovelbotulinumneurotoxinasubtype6 AT ericajohnson isolationandcharacterizationofthenovelbotulinumneurotoxinasubtype6 AT sabinepellett isolationandcharacterizationofthenovelbotulinumneurotoxinasubtype6
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Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6

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