Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium Botulinum Expression System

Overview

Journal Appl Environ Microbiol

Specialties Environmental Health
Microbiology

Date 2014 Sep 21

PMID 25239905

Citations 17

Authors

Marite Bradshaw

William H Tepp

Regina C M Whitemarsh

Sabine Pellett

Eric A Johnson

Affiliations

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Abstract

Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

Citing Articles

Expression of Recombinant Clostridial Neurotoxin by .

Gregg B, Gupta S, Tepp W, Pellett S Microorganisms. 2025; 12(12.

PMID: 39770813 PMC: 11678509. DOI: 10.3390/microorganisms12122611.

Potency Evaluations of Recombinant Botulinum Neurotoxin A1 Mutants Designed to Reduce Toxicity.

Viravathana P, Tepp W, Bradshaw M, Przedpelski A, Barbieri J, Pellett S Int J Mol Sci. 2024; 25(16).

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Botulinum neurotoxin X lacks potency in mice and in human neurons.

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PMID: 38347673 PMC: 10936432. DOI: 10.1128/mbio.03106-23.

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Gupta S, Pellett S Toxins (Basel). 2023; 15(9).

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Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain.

Tepp W, Bradshaw M, Gardner A, Kaufman R, Barbieri J, Pellett S Int J Mol Sci. 2023; 24(6).

PMID: 36982762 PMC: 10055998. DOI: 10.3390/ijms24065690.

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