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Special Issue "Advances in Clostridial and Related Neurotoxins 2.0"

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: 15 March 2024 | Viewed by 8399

Special Issue Editor

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. The 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 the 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 a 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 (6 papers)

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Research

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21 pages, 5985 KiB  
Article
Crystal Structure of the Catalytic Domain of a Botulinum Neurotoxin Homologue from Enterococcus faecium: Potential Insights into Substrate Recognition
Int. J. Mol. Sci. 2023, 24(16), 12721; https://doi.org/10.3390/ijms241612721 - 12 Aug 2023
Viewed by 661
Abstract
Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn2+-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several [...] Read more.
Clostridium botulinum neurotoxins (BoNTs) are the most potent toxins known, causing the deadly disease botulism. They function through Zn2+-dependent endopeptidase cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, preventing vesicular fusion and subsequent neurotransmitter release from motor neurons. Several serotypes of BoNTs produced by Clostridium botulinum (BoNT/A-/G and/X) have been well-characterised over the years. However, a BoNT-like gene (homologue of BoNT) was recently identified in the non-clostridial species, Enterococcus faecium, which is the leading cause of hospital-acquired multi-drug resistant infections. Here, we report the crystal structure of the catalytic domain of a BoNT homologue from Enterococcus faecium (LC/En) at 2.0 Å resolution. Detailed structural analysis in comparison with the full-length BoNT/En AlphaFold2-predicted structure, LC/A (from BoNT/A), and LC/F (from BoNT/F) revealed putative subsites and exosites (including loops 1–5) involved in recognition of LC/En substrates. LC/En also appears to possess a conserved autoproteolytic cleavage site whose function is yet to be established. Full article
(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins 2.0)
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14 pages, 1129 KiB  
Article
Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain
Int. J. Mol. Sci. 2023, 24(6), 5690; https://doi.org/10.3390/ijms24065690 - 16 Mar 2023
Cited by 1 | Viewed by 924
Abstract
Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) [...] Read more.
Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a β-strand peptide (556–564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the β-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 β-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 β-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C β-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C β-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563. Full article
(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins 2.0)
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30 pages, 2591 KiB  
Article
Transformation of a Metal Chelate into a “Catch and Anchor” Inhibitor of Botulinum A Protease
Int. J. Mol. Sci. 2023, 24(5), 4303; https://doi.org/10.3390/ijms24054303 - 21 Feb 2023
Cited by 2 | Viewed by 1205
Abstract
Targeting the botulinum neurotoxin light chain (LC) metalloprotease using small-molecule metal chelate inhibitors is a promising approach to counter the effects of the lethal toxin. However, to overcome the pitfalls associated with simple reversible metal chelate inhibitors, it is crucial to investigate alternative [...] Read more.
Targeting the botulinum neurotoxin light chain (LC) metalloprotease using small-molecule metal chelate inhibitors is a promising approach to counter the effects of the lethal toxin. However, to overcome the pitfalls associated with simple reversible metal chelate inhibitors, it is crucial to investigate alternative scaffolds/strategies. In conjunction with Atomwise Inc., in silico and in vitro screenings were conducted, yielding a number of leads, including a novel 9-hydroxy-4H-pyrido [1,2-a]pyrimidin-4-one (PPO) scaffold. From this structure, an additional series of 43 derivatives were synthesized and tested, resulting in a lead candidate with a Ki of 150 nM in a BoNT/A LC enzyme assay and 17 µM in a motor neuron cell-based assay. These data combined with structure-activity relationship (SAR) analysis and docking led to a bifunctional design strategy, which we termed “catch and anchor” for the covalent inhibition of BoNT/A LC. Kinetic evaluation was conducted on structures prepared from this catch and anchor campaign, providing kinact/Ki values, and rationale for inhibition seen. Covalent modification was validated through additional assays, including an FRET endpoint assay, mass spectrometry, and exhaustive enzyme dialysis. The data presented support the PPO scaffold as a novel candidate for targeted covalent inhibition of BoNT/A LC. Full article
(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins 2.0)
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15 pages, 2650 KiB  
Article
Lasting Peripheral and Central Effects of Botulinum Toxin Type A on Experimental Muscle Hypertonia in Rats
Int. J. Mol. Sci. 2022, 23(19), 11626; https://doi.org/10.3390/ijms231911626 - 01 Oct 2022
Cited by 2 | Viewed by 1337
Abstract
Recent animal experiments suggested that centrally transported botulinum toxin type A (BoNT-A) might reduce an abnormal muscle tone, though with an unknown contribution to the dominant peripheral muscular effect observed clinically. Herein, we examined if late BoNT-A antispastic actions persist due to possible [...] Read more.
Recent animal experiments suggested that centrally transported botulinum toxin type A (BoNT-A) might reduce an abnormal muscle tone, though with an unknown contribution to the dominant peripheral muscular effect observed clinically. Herein, we examined if late BoNT-A antispastic actions persist due to possible central toxin actions in rats. The early effect of intramuscular (i.m.) BoNT-A (5, 2 and 1 U/kg) on a reversible tetanus toxin (TeNT)-induced calf muscle spasm was examined 7 d post-TeNT and later during recovery from flaccid paralysis (TeNT reinjected on day 49 post-BoNT-A). Lumbar intrathecal (i.t.) BoNT-A–neutralizing antiserum was used to discriminate the transcytosis-dependent central toxin action of 5 U/kg BoNT-A. BoNT-A-truncated synaptosomal-associated protein 25 immunoreactivity was examined in the muscles and spinal cord at day 71 post-BoNT-A. All doses (5, 2 and 1 U/kg) induced similar antispastic actions in the early period (days 1–14) post-BoNT-A. After repeated TeNT, only the higher two doses prevented the muscle spasm and associated locomotor deficit. Central trans-synaptic activity contributed to the late antispastic effect of 5 U/kg BoNT-A. Ongoing BoNT-A enzymatic activity was present in both injected muscle and the spinal cord. These observations suggest that the treatment duration in sustained or intermittent muscular hyperactivity might be maintained by higher doses and combined peripheral and central BoNT-A action. Full article
(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins 2.0)
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11 pages, 4363 KiB  
Article
Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility
Int. J. Mol. Sci. 2022, 23(17), 9620; https://doi.org/10.3390/ijms23179620 - 25 Aug 2022
Cited by 2 | Viewed by 1647
Abstract
Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at [...] Read more.
Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT’s high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6 (crystal form II) where a large ‘hinge motion’ between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 (crystal form I), reveals the degree of conformational flexibility exhibited by HC/A6. Full article
(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins 2.0)
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Review

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17 pages, 4238 KiB  
Review
Pathology and Pathogenesis of Brain Lesions Produced by Clostridium perfringens Type D Epsilon Toxin
Int. J. Mol. Sci. 2022, 23(16), 9050; https://doi.org/10.3390/ijms23169050 - 12 Aug 2022
Cited by 5 | Viewed by 1943
Abstract
Clostridium perfringens type D epsilon toxin (ETX) produces severe, and frequently fatal, neurologic disease in ruminant livestock. The disorder is of worldwide distribution and, although vaccination has reduced its prevalence, ETX still causes substantial economic loss in livestock enterprises. The toxin is produced [...] Read more.
Clostridium perfringens type D epsilon toxin (ETX) produces severe, and frequently fatal, neurologic disease in ruminant livestock. The disorder is of worldwide distribution and, although vaccination has reduced its prevalence, ETX still causes substantial economic loss in livestock enterprises. The toxin is produced in the intestine as a relatively inactive prototoxin, which is subsequently fully enzymatically activated to ETX. When changed conditions in the intestinal milieu, particularly starch overload, favor rapid proliferation of this clostridial bacterium, large amounts of ETX can be elaborated. When sufficient toxin is absorbed from the intestine into the systemic circulation and reaches the brain, two neurologic syndromes can develop from this enterotoxemia. If the brain is exposed to large amounts of ETX, the lesions are fundamentally vasculocentric. The neurotoxin binds to microvascular endothelial receptors and other brain cells, the resulting damage causing increased vascular permeability and extravasation of plasma protein and abundant fluid into the brain parenchyma. While plasma protein, particularly albumin, pools largely perivascularly, the vasogenic edema becomes widely distributed in the brain, leading to a marked rise in intracranial pressure, coma, sometimes cerebellar herniation, and, eventually, often death. When smaller quantities of ETX are absorbed into the bloodstream, or livestock are partially immune, a more protracted clinical course ensues. The resulting brain injury is characterized by bilaterally symmetrical necrotic foci in certain selectively vulnerable neuroanatomic sites, termed focal symmetrical encephalomalacia. ETX has also been internationally listed as a potential bioterrorism agent. Although there are no confirmed human cases of ETX intoxication, the relatively wide species susceptibility to this toxin and its high toxicity mean it is likely that human populations would also be vulnerable to its neurotoxic actions. While the pathogenesis of ETX toxicity in the brain is incompletely understood, the putative mechanisms involved in neural lesion development are discussed. Full article
(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins 2.0)
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