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Advances in Clostridial and Related Neurotoxins, 3rd Edition
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Advances in Clostridial and Related Neurotoxins, 3rd Edition

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: 30 October 2024 | Viewed by 689

Special Issue Editor

Dr. Sabine Pellett

E-Mail Website
Guest Editor
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

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-pharmaceutical agents. 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 biopharmaceuticals. 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

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Published Papers (1 paper)

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Research

18 pages, 2471 KiB  
Article
Potency Evaluations of Recombinant Botulinum Neurotoxin A1 Mutants Designed to Reduce Toxicity
by Polrit Viravathana, William H. Tepp, Marite Bradshaw, Amanda Przedpelski, Joseph T. Barbieri and Sabine Pellett
Int. J. Mol. Sci. 2024, 25(16), 8955; https://doi.org/10.3390/ijms25168955 - 17 Aug 2024
Viewed by 528
Abstract
Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2–3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold–12.5-million-fold) versus wild-type BoNT/A1, leading to their current exclusion from the Federal [...] Read more.
Recombinant mutant holotoxin BoNTs (rBoNTs) are being evaluated as possible vaccines against botulism. Previously, several rBoNTs containing 2–3 amino acid mutations in the light chain (LC) showed significant decreases in toxicity (2.5-million-fold–12.5-million-fold) versus wild-type BoNT/A1, leading to their current exclusion from the Federal Select Agent list. In this study, we added four additional mutations in the receptor-binding domain, translocation domain, and enzymatic cleft to further decrease toxicity, creating 7M rBoNT/A1. Due to poor expression in E. coli, 7M rBoNT/A1 was produced in an endogenous C. botulinum expression system. This protein had higher residual toxicity (LD50: 280 ng/mouse) than previously reported for the catalytically inactive rBoNT/A1 containing only three of the mutations (>10 µg/mouse). To investigate this discrepancy, several additional rBoNT/A1 constructs containing individual sets of amino acid substitutions from 7M rBoNT/A1 and related mutations were also endogenously produced. Similarly to endogenously produced 7M rBoNT/A1, all of the endogenously produced mutants had ~100–1000-fold greater toxicity than what was reported for their original heterologous host counterparts. A combination of mutations in multiple functional domains resulted in a greater but not multiplicative reduction in toxicity. This report demonstrates the impact of production systems on residual toxicity of genetically inactivated rBoNTs. Full article
(This article belongs to the Special Issue Advances in Clostridial and Related Neurotoxins, 3rd Edition)
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Figure 1

Figure 1
<p>Design of 7M BoNT/A1. The mutations in 7M rBoNT/A1 are based on several different published mutations in clostridial neurotoxins combined into a single BoNT/A1 holotoxin. A single-point mutation in the ganglioside binding domain (W1266A), as well as translocation domain (K759A), was introduced into the ~100 kDa heavy chain [<a href="#B39-ijms-25-08955" class="html-bibr">39</a>,<a href="#B42-ijms-25-08955" class="html-bibr">42</a>]. Two sets of mutations were placed in the metalloprotease light chain: a double mutation (L175A and D370A) in the enzymatic cleft targeting hydrolytic activity and three-point mutations (E224Q, R363A, and Y366F) introduced into the zinc coordination motif, which has shown to significantly abrogate toxin activity and potency in an <span class="html-italic">E. coli</span>-produced rBoNT/A1<sup>E224Q,R363A,Y366F</sup> [<a href="#B30-ijms-25-08955" class="html-bibr">30</a>,<a href="#B40-ijms-25-08955" class="html-bibr">40</a>]. (<b>A</b>) Specific mutation(s) that make up the 7M BoNT/A1 design, including their target and published origin. (<b>B</b>) Overlay of 7M BoNT/A1 (magenta) vs. wt BoNT/A1 (green). The altered amino acid residues in 7M rBoNT/A1 are shown in cyan spheres. The zinc ion of the wild-type BoNT/A1 LC is shown in red. The 7M BoNT/A1 amino acid sequence was inputted into Phyre 2 [<a href="#B43-ijms-25-08955" class="html-bibr">43</a>] to generate a structural model using intensive mode. This structural model of 7M BoNT/A1 crafted in Phyre2 [<a href="#B43-ijms-25-08955" class="html-bibr">43</a>] was subsequently superimposed with the wild-type BoNT/A1 (PDB ID 2NYY) using PyMol [<a href="#B41-ijms-25-08955" class="html-bibr">41</a>]. PyMOL Molecular Graphics System, Version 4.6 Schrödinger, LLC (New York, NY, USA).</p> Full article ">Figure 2
<p>The purity and biological activity of <span class="html-italic">Clostridium</span> produced wild-type and recombinant 7M rBoNT/A1. Wild-type BoNT/A1 was produced in <span class="html-italic">C. botulinum</span> in strain Hall A-hyper, while the mutant 7M rBoNT/A1 was produced in <span class="html-italic">C. botulinum</span> strain Hall A-hyper/tox<sup>−</sup>. Both were purified using biochemical methods. 7M rBoNT/A1 was successfully produced in an endogenous clostridial expression system, which also produces all BoNT/A complexing proteins but no wild-type toxin. The 7M rBoNT/A1 was also successfully synthesized and purified without the use of purification tags or additional modifications [<a href="#B44-ijms-25-08955" class="html-bibr">44</a>]. (<b>A</b>) Coomassie-stained SDS-PAGE gels of reduced (R) and non-reduced (NR) samples of purified wild-type and 7M rBoNT/A1. (<b>B</b>) Specific toxicity of wild-type BoNT/A1 and 7M rBoNT/A1 in mice. (<b>C</b>) Graphic representation of Western blot data analyzing potency of wt BoNT/A1 and 7M rBoNT/A1 in primary rat spinal cord cells. The cultured neurons were exposed to serial dilutions of the indicated toxins for 48 h, and the percent SNAP25 cleavage was determined in cell lysates by Western blot, using an anti-SNAP-25 antibody that equally detects both uncleaved and cleaved SNAP-25, and densitometry. Values were then plotted in GraphPad Prism Version 9 (<span class="html-italic">n</span> ≥ 3), and the resulting graphs and EC50 (Half Maximal Effective Concentration) values were derived from a nonlinear four-parameter regression curve fit. A comparative EC50 for 7M rBoNT/A1 could not be determined (N.D.) since 100% cleavage was not achieved under experimental conditions. (<b>D</b>) Representative Western blots of the primary RSC cell-based Assays. u: uncleaved SNAP-25, c: cleaved SNAP-25. All Western blots used to determine the EC50 are shown in <a href="#app1-ijms-25-08955" class="html-app">Supplementary Figure S1</a>.</p> Full article ">Figure 3
<p>The purity and biological activity of <span class="html-italic">Clostridium</span> produced rBoNT/A1<sup>K759A</sup>. The mutant BoNT/A1<sup>K759A</sup> was produced in <span class="html-italic">C. botulinum</span> strain Hall A-hyper/tox<sup>−</sup> and purified using biochemical methods. (<b>A</b>) Coomassie-stained SDS-PAGE gels of reduced (R) and non-reduced (NR) purified samples of rBoNT/A1<sup>K759A</sup>. (<b>B</b>) Specific toxicity of this rBoNT/A1<sup>K759A</sup> in mice, compared to wt BoNT/A1. (<b>C</b>) Graphic representation of Western blot data analyzing potency of rBoNT/A1<sup>K759A</sup> relative to wt BoNT/A1 and 7M BoNT/A1 in primary rat spinal cord cells. The cultured neurons were exposed to serial dilutions of the indicated toxins for 48 h, and the percent SNAP25 cleavage was determined in cell lysates by Western blot, using an anti-SNAP-25 antibody that equally detects both uncleaved and cleaved SNAP-25, and densitometry. Values were then plotted in GraphPad Prism Version 9 (<span class="html-italic">n</span> ≥ 3), and the resulting graphs and EC50 (Half Maximal Effective Concentration) values were derived from a nonlinear four-parameter regression curve fit. (<b>D</b>) Representative Western blots of the primary RSC cell-based Assays. u, uncleaved SNAP-25; c, cleaved SNAP-25. All Western blots used to determine the EC50 are shown in the <a href="#app1-ijms-25-08955" class="html-app">Supplementary Figure S2</a>.</p> Full article ">Figure 4
<p>The purity and biological activity of <span class="html-italic">Clostridium</span>-produced rBoNT/A1 with mutations targeting the binding capabilities of the HC. All mutant rBoNT/A1s were produced in <span class="html-italic">C. botulinum</span> strain Hall A-hyper/tox<sup>−</sup> and purified using biochemical methods. (<b>A</b>) Coomassie-stained SDS-PAGE gels of reduced (R) and non-reduced (NR) samples of purified rBoNT/A1<sup>W1266A</sup> and rBoNT/A1<sup>G1292R</sup>. (<b>B</b>) Specific toxicity of both mutant holotoxins in mice, compared to wt BoNT/A1. (<b>C</b>) Graphic representation of Western blot data analyzing potency of rBoNT/A1<sup>W1266A</sup> and rBoNT/A1<sup>G1292R</sup> relative to wt BoNT/A1 and 7M BoNT/A1 in primary rat spinal cord cells. The cultured neurons were exposed to serial dilutions of the indicated toxins for 48 h, and the percent SNAP25 cleavage was determined in cell lysates by Western blot, using an anti-SNAP-25 antibody equally detects both uncleaved and cleaved SNAP-25, and densitometry. Values were then plotted in GraphPad Prism Version 9 (<span class="html-italic">n</span> ≥ 3), and the resulting graphs and EC50 (Half Maximal Effective Concentration) values were derived from a nonlinear four-parameter regression curve fit. (<b>D</b>) Representative Western blots of the primary RSC cell-based Assays. u, uncleaved SNAP-25; c, cleaved SNAP-25. All Western blots used to determine the EC50 are shown in <a href="#app1-ijms-25-08955" class="html-app">Supplementary Figure S3</a>.</p> Full article ">Figure 5
<p>The purity and biological activity of all <span class="html-italic">Clostridium</span>-produced rBoNT/A1 with mutations targeting the different activities of the BoNT/A1 LC. All mutant rBoNT/A1s were produced in <span class="html-italic">C. botulinum</span> strain Hall A-hyper/tox<sup>−</sup> and purified using biochemical methods. (<b>A</b>) Coomassie-stained SDS-PAGE gels of reduced (R) and non-reduced (NR) samples of purified rBoNT/A1<sup>L175A,D370A</sup> that abrogate the LC enzymatic activity, as well as rBoNT/A1<sup>E224Q,R363A,Y366F</sup> and ciBoNT/A1, which abolish the zinc ion coordination of the LC metalloprotease. (<b>B</b>) Specific toxicity of all mutant holotoxins in mice, compared to wt BoNT/A1. (<b>C</b>) Graphic representation of Western blot data analyzing potency of rBoNT/A1<sup>L175A,D370A</sup>, rBoNT/A1<sup>E224Q,R363A,Y366F</sup>, and rBoNT/A1<sup>H223A,E224Q,H227A</sup> (ciBoNT/A1) relative to wt BoNT/A1 and 7M BoNT/A1 in primary rat spinal cord cells. The cultured neurons were exposed to serial dilutions of the indicated toxins for 48 h, and the percent SNAP25 cleavage was determined in cell lysates by Western blot, using an anti-SNAP-25 antibody that equally detects both uncleaved and cleaved SNAP-25, and densitometry. Values were then plotted in GraphPad Prism Version 9 (<span class="html-italic">n</span> ≥ 3), and the resulting graphs and EC50 (Half Maximal Effective Concentration) values were derived from a nonlinear four-parameter regression curve fit. A comparative EC50 for rBoNT/A1<sup>E224Q,R363A,Y366F</sup> and rBoNT/A1<sup>H223A,E224Q,H227A</sup> (ciBoNT/A1) could not be determined (N.D.) since 100% cleavage was not achieved under experimental conditions. (<b>D</b>) Representative Western blots of the primary RSC cell-based assays. u, uncleaved SNAP-25; c, cleaved SNAP-25. All Western blots used to determine the EC50 are shown in <a href="#app1-ijms-25-08955" class="html-app">Supplementary Figure S4</a>.</p> Full article ">
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