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Special Issue "Novel BoNTs and Toxin Engineering"

A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Bacterial Toxins".

Deadline for manuscript submissions: closed (31 May 2018)

Special Issue Editor

Guest Editor
Dr. Sabine Pellett

UW-Madison, Department of Bacteriology, Madison, WI 53706, USA
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Special Issue Information

Dear Colleagues,

Botulinum Neurotoxins (BoNTs), the causative agents of the potentially lethal vertebrate disease botulism, comprise a large and expanding family of protein toxins produced by various bacterial strains of the genus Clostridium. BoNTs are significant as disease-causing agents, potential bioterrorist agents, and as unique, long-lasting, and widely used bio-pharmaceuticals. Currently, BoNTs are categorized into seven immunologically distinct serotypes with several subtypes within each serotype. However, in recent years, discoveries of novel BoNTs, as well as potential BoNT homologues in other organisms, have challenged this categorization and expanded the family of BoNTs. While novel BoNTs are continually being identified by sequencing, most have not been purified and functionally characterized. Further identification and characterizations of novel and known BoNTs will yield insight into the evolutionary forces driving the diversity of this protein toxin family and potentially reveal as yet unknown pharmacologic properties of BoNTs, with the potential to lead to novel or improved BoNT based bio-pharmaceuticals. Furthermore, genetic methods now allow for construction of recombinant and chimeric BoNTs, enabling directed engineering of BoNTs with defined amino acid or functional domain substitutions. Combined with ongoing structural analyses, these studies will lead to a deeper understanding of the molecular mechanisms underlying the toxicity and pharmacologic potential of the large family of BoNTs. Both approaches, exploring novel BoNTs and recombinant studies, are exciting avenues of research with the potential to open the doors to unlocking the underlying molecular and evolutionary mechanisms of the high potency of BoNTs, eventually leading to improved safety approaches, countermeasures development, and novel pharmaceuticals and pharmaceutical applications. 

Dr. Sabine Pellett
Guest Editor

Manuscript Submission Information

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Keywords

  • Botulinum Neurotoxin
  • BoNT
  • novel
  • recombinant
  • derivative
  • Clostridium botulinum

Published Papers (7 papers)

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Research

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Open AccessArticle Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58–K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities
Received: 14 June 2018 / Revised: 23 July 2018 / Accepted: 30 July 2018 / Published: 1 August 2018
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Abstract
In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also
[...] Read more.
In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here, we report the primary structure of a ninth subtype of BoNT/F. Its amino-acid sequence diverges by at least 8.4% at the holotoxin and 13.4% at the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6, and vary by up to a factor of eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide, the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype. Full article
(This article belongs to the Special Issue Novel BoNTs and Toxin Engineering)
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Open AccessArticle Purification and Characterization of Recombinant Botulinum Neurotoxin Serotype FA, Also Known as Serotype H
Received: 17 April 2018 / Revised: 4 May 2018 / Accepted: 8 May 2018 / Published: 11 May 2018
Cited by 2 | PDF Full-text (1912 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here
[...] Read more.
We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here we report purification and characterization of enzymatically active, and of inactive nontoxic, recombinant forms of BoNT/FA as tractable alternatives to purifying this neurotoxin from native Clostridium botulinum. BoNT/FA cleaves the same intracellular target proteins as BoNT/F1 and other F serotype BoNTs; the intracellular targets are vesicle associated membrane proteins (VAMP) 1, 2 and 3. BoNT/FA cleaves the same site in VAMP-2 as BoNT/F5, which is different from the cleavage site of other F serotype BoNTs. BoNT/FA has slower enzyme kinetics than BoNT/F1 in a cell-free protease assay and is less potent at inhibiting ex vivo nerve-stimulated skeletal muscle contraction. In contrast, BoNT/FA is more potent at inhibiting neurotransmitter release from cultured neurons. Full article
(This article belongs to the Special Issue Novel BoNTs and Toxin Engineering)
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Review

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Open AccessReview Engineering Botulinum Toxins to Improve and Expand Targeting and SNARE Cleavage Activity
Received: 25 May 2018 / Revised: 29 June 2018 / Accepted: 1 July 2018 / Published: 4 July 2018
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Abstract
Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar
[...] Read more.
Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar multi-domain structure at the molecular level. In nature, BoNTs are encoded by DNA in producing clostridial bacteria and, as such, are amenable to recombinant production through insertion of the coding DNA into other bacterial species. This, in turn, creates possibilities for protein engineering. Here, we review the production of BoNTs by the natural host and also recombinant production approaches utilised in the field. Applications of recombinant BoNT-production include the generation of BoNT-derived domain fragments, the creation of novel BoNTs with improved performance and enhanced therapeutic potential, as well as the advancement of BoNT vaccines. In this article, we discuss site directed mutagenesis, used to affect the biological properties of BoNTs, including approaches to alter their binding to neurons and to alter the specificity and kinetics of substrate cleavage. We also discuss the target secretion inhibitor (TSI) platform, in which the neuronal binding domain of BoNTs is substituted with an alternative cellular ligand to re-target the toxins to non-neuronal systems. Understanding and harnessing the potential of the biological diversity of natural BoNTs, together with the ability to engineer novel mutations and further changes to the protein structure, will provide the basis for increasing the scope of future BoNT-based therapeutics. Full article
(This article belongs to the Special Issue Novel BoNTs and Toxin Engineering)
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Open AccessReview Light Chain Diversity among the Botulinum Neurotoxins
Received: 1 June 2018 / Revised: 19 June 2018 / Accepted: 21 June 2018 / Published: 2 July 2018
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Abstract
Botulinum neurotoxins (BoNT) are produced by several species of clostridium. There are seven immunologically unique BoNT serotypes (A–G). The Centers for Disease Control classifies BoNTs as ‘Category A’ select agents and are the most lethal protein toxins for humans. Recently, BoNT-like proteins have
[...] Read more.
Botulinum neurotoxins (BoNT) are produced by several species of clostridium. There are seven immunologically unique BoNT serotypes (A–G). The Centers for Disease Control classifies BoNTs as ‘Category A’ select agents and are the most lethal protein toxins for humans. Recently, BoNT-like proteins have also been identified in several non-clostridia. BoNTs are di-chain proteins comprised of an N-terminal zinc metalloprotease Light Chain (LC) and a C-terminal Heavy Chain (HC) which includes the translocation and receptor binding domains. The two chains are held together by a disulfide bond. The LC cleaves Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The cleavage of SNAREs inhibits the fusion of synaptic vesicles to the cell membrane and the subsequent release of acetylcholine, which results in flaccid paralysis. The LC controls the catalytic properties and the duration of BoNT action. This review discusses the mechanism for LC catalysis, LC translocation, and the basis for the duration of LC action. Understanding these properties of the LC may expand the applications of BoNT as human therapies. Full article
(This article belongs to the Special Issue Novel BoNTs and Toxin Engineering)
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Open AccessReview Engineering of Botulinum Neurotoxins for Biomedical Applications
Received: 2 May 2018 / Revised: 1 June 2018 / Accepted: 5 June 2018 / Published: 6 June 2018
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Abstract
Botulinum neurotoxins (BoNTs) have been used as therapeutic agents in the clinical treatment of a wide array of neuromuscular and autonomic neuronal transmission disorders. These toxins contain three functional domains that mediate highly specific neuronal cell binding, internalization and cytosolic delivery of proteolytic
[...] Read more.
Botulinum neurotoxins (BoNTs) have been used as therapeutic agents in the clinical treatment of a wide array of neuromuscular and autonomic neuronal transmission disorders. These toxins contain three functional domains that mediate highly specific neuronal cell binding, internalization and cytosolic delivery of proteolytic enzymes that cleave proteins integral to the exocytosis of neurotransmitters. The exceptional cellular specificity, potency and persistence within the neuron that make BoNTs such effective toxins, also make them attractive models for derivatives that have modified properties that could potentially expand their therapeutic repertoire. Advances in molecular biology techniques and rapid DNA synthesis have allowed a wide variety of novel BoNTs with alternative functions to be assessed as potential new classes of therapeutic drugs. This review examines how the BoNTs have been engineered in an effort to produce new classes of therapeutic molecules to address a wide array of disorders. Full article
(This article belongs to the Special Issue Novel BoNTs and Toxin Engineering)
Open AccessReview Novel Botulinum Neurotoxins: Exploring Underneath the Iceberg Tip
Received: 15 April 2018 / Revised: 5 May 2018 / Accepted: 8 May 2018 / Published: 10 May 2018
Cited by 7 | PDF Full-text (829 KB) | HTML Full-text | XML Full-text
Abstract
Botulinum neurotoxins (BoNTs), the etiological agents of botulism, are the deadliest toxins known to humans. Yet, thanks to their biological and toxicological features, BoNTs have become sophisticated tools to study neuronal physiology and valuable therapeutics for an increasing number of human disorders. BoNTs
[...] Read more.
Botulinum neurotoxins (BoNTs), the etiological agents of botulism, are the deadliest toxins known to humans. Yet, thanks to their biological and toxicological features, BoNTs have become sophisticated tools to study neuronal physiology and valuable therapeutics for an increasing number of human disorders. BoNTs are produced by multiple bacteria of the genus Clostridium and, on the basis of their different immunological properties, were classified as seven distinct types of toxin. BoNT classification remained stagnant for the last 50 years until, via bioinformatics and high-throughput sequencing techniques, dozens of BoNT variants, novel serotypes as well as BoNT-like toxins within non-clostridial species have been discovered. Here, we discuss how the now “booming field” of botulinum neurotoxin may shed light on their evolutionary origin and open exciting avenues for future therapeutic applications. Full article
(This article belongs to the Special Issue Novel BoNTs and Toxin Engineering)
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Other

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Open AccessOpinion Botulinum Neurotoxin Diversity from a Gene-Centered View
Received: 13 June 2018 / Revised: 24 July 2018 / Accepted: 30 July 2018 / Published: 1 August 2018
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Abstract
Botulinum neurotoxins (BoNTs) rank amongst the most potent toxins known. The factors responsible for the emergence of the many known and yet unknown BoNT variants remain elusive. It also remains unclear why anaerobic bacteria that are widely distributed in our environment and normally
[...] Read more.
Botulinum neurotoxins (BoNTs) rank amongst the most potent toxins known. The factors responsible for the emergence of the many known and yet unknown BoNT variants remain elusive. It also remains unclear why anaerobic bacteria that are widely distributed in our environment and normally do not pose a threat to humans, produce such deadly toxins. Even the possibility of accidental toxicity to humans has not been excluded. Here, I review the notion that BoNTs may have specifically evolved to target vertebrates. Considering the extremely complex molecular architecture of the toxins, which enables them to reach the bloodstream, to recognize and enter neurons, and to block neurotransmitter release, it seems highly unlikely that BoNT toxicity to vertebrates is a coincidence. The carcass–maggot cycle provides a plausible explanation for a natural role of the toxins: to enable mass reproduction of bacteria, spores, and toxins, using toxin-unaffected invertebrates, such as fly maggots, as the vectors. There is no clear correlation between toxigenicity and a selective advantage of clostridia in their natural habitat. Possibly, non-toxigenic strains profit from carcasses resulting from the action of toxigenic strains. Alternatively, a gene-centered view of toxin evolution would also explain this observation. Toxin-coding mobile genetic elements may have evolved as selfish genes, promoting their own propagation, similar to commensal viruses, using clostridia and other bacteria as the host. Research addressing the role of BoNTs in nature and the origin of toxin variability goes hand in hand with the identification of new toxin variants and the design of improved toxin variants for medical applications. These research directions may also reveal yet unknown natural antidotes against these extremely potent neurotoxins. Full article
(This article belongs to the Special Issue Novel BoNTs and Toxin Engineering)
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