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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Toxicology".

Deadline for manuscript submissions: closed (30 April 2022) | Viewed by 15832

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Dr. Sabine Pellett

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Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA
Interests: botulinum toxins; food safety; plasmids; transposons; bacteriophages
Special Issues, Collections and Topics in MDPI journals

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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 characterization of novel and known BoNTs will yield insights 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 the 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 door to unlocking the underlying molecular and evolutionary mechanisms of the high potency of BoNTs, eventually leading to improved safety approaches, countermeasure development, and novel pharmaceuticals and pharmaceutical applications.

Dr. Sabine Pellett
Guest Editor

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Keywords

Botulinum Neurotoxin
BoNT
toxicity
molecular mechanisms
recombinant
derivative
Clostridium botulinum

Published Papers (7 papers)

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Editorial

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4 pages, 355 KiB

Open AccessEditorial

Advances in Clostridial and Related Neurotoxins

by Sabine Pellett

Int. J. Mol. Sci. 2022, 23(22), 14076; https://doi.org/10.3390/ijms232214076 - 15 Nov 2022

Cited by 1 | Viewed by 886

Abstract

The huge advances in genomics and molecular biology in the past two decades have made now an exciting time to study bacterial toxins, in particular, the most potent bacterial toxin known to humankind, botulinum neurotoxins (BoNTs) [...] Full article

(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins)

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Research

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15 pages, 3044 KiB

Open AccessArticle

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody

by Federico Fabris, Petra Šoštarić, Ivica Matak, Thomas Binz, Anna Toffan, Morena Simonato, Cesare Montecucco, Marco Pirazzini and Ornella Rossetto

Int. J. Mol. Sci. 2022, 23(8), 4355; https://doi.org/10.3390/ijms23084355 - 14 Apr 2022

Cited by 5 | Viewed by 2578

Abstract

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo. Full article

(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins)

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16 pages, 2969 KiB

Open AccessArticle

Novel Putative Transposable Element Associated with the Subtype E5 Botulinum Toxin Gene Cluster of Neurotoxigenic Clostridium butyricum Type E Strains from China

by Tao Li, Nianzhi Ning, Angelo Iacobino, Liangyan Zhang, Hui Wang and Giovanna Franciosa

Int. J. Mol. Sci. 2022, 23(2), 906; https://doi.org/10.3390/ijms23020906 - 14 Jan 2022

Cited by 1 | Viewed by 1770

Abstract

Previously, a whole-genome comparison of three Clostridium butyricum type E strains from Italy and the United States with different C. botulinum type E strains indicated that the bont/e gene might be transferred between the two clostridia species through transposition. However, transposable elements (TEs) have never been identified close to the bont/e gene. Herein, we report the whole genome sequences for four neurotoxigenic C. butyricum type E strains that originated in China. An analysis of the obtained genome sequences revealed the presence of a novel putative TE upstream of the bont/e gene in the genome of all four strains. Two strains of environmental origin possessed an additional copy of the putative TE in their megaplasmid. Similar putative TEs were found in the megaplasmids and, less frequently, in the chromosomes of several C. butyricum strains, of which two were neurotoxigenic C. butyricum type E strains, and in the chromosome of a single C. botulinum type E strain. We speculate that the putative TE might potentially transpose the bont/e gene at the intracellular and inter-cellular levels. However, the occasional TE occurrence in the clostridia genomes might reflect rare transposition events. Full article

(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins)

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15 pages, 24740 KiB

Open AccessArticle

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane

by Alexander Gardner, William H. Tepp, Marite Bradshaw, Joseph T. Barbieri and Sabine Pellett

Int. J. Mol. Sci. 2021, 22(20), 11115; https://doi.org/10.3390/ijms222011115 - 15 Oct 2021

Cited by 3 | Viewed by 1576

Abstract

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1–A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy. Full article

(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins)

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17 pages, 3211 KiB

Open AccessArticle

Selective Expression of a SNARE-Cleaving Protease in Peripheral Sensory Neurons Attenuates Pain-Related Gene Transcription and Neuropeptide Release

by Wanzhi Wang, Miaomiao Kong, Yu Dou, Shanghai Xue, Yang Liu, Yinghao Zhang, Weiwei Chen, Yanqing Li, Xiaolong Dai, Jianghui Meng and Jiafu Wang

Int. J. Mol. Sci. 2021, 22(16), 8826; https://doi.org/10.3390/ijms22168826 - 17 Aug 2021

Cited by 9 | Viewed by 2619

Abstract

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief. Full article

(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins)

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15 pages, 11001 KiB

Open AccessArticle

Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E

by Alon Ben David, Ada Barnea, Eran Diamant, Eyal Dor, Arieh Schwartz, Amram Torgeman and Ran Zichel

Int. J. Mol. Sci. 2021, 22(16), 8577; https://doi.org/10.3390/ijms22168577 - 09 Aug 2021

Cited by 5 | Viewed by 2514

Abstract

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures. Full article

(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins)

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12 pages, 2403 KiB

Open AccessArticle

Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E

by Geoffrey Masuyer, Jonathan R. Davies and Pål Stenmark

Int. J. Mol. Sci. 2021, 22(15), 8315; https://doi.org/10.3390/ijms22158315 - 02 Aug 2021

Cited by 5 | Viewed by 2896

Abstract

The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228–1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides. Full article

(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins)

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