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

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.

FIG 1

Western blot analyses of botulinum neurotoxin expression in C. botulinum wild-type strain Hall A-hyper (BoNT/A1) and in four independent nontoxigenic mutant clones using polyclonal antibodies raised against serotype A1 BoNT. Purified botulinum neurotoxin (reduced and nonreduced) isolated from strain Hall A-hyper was used as a standard; all other samples were reduced. scBoNT, single-chain botulinum neurotoxin; LC/BoNT, botulinum neurotoxin light chain; HC/BoNT, botulinum neurotoxin heavy chain; wt, wild type; tox⁻, nontoxigenic mutants.

FIG 2

rBoNT/A4 (A) and BoNT/A4-His (B and C) expression in C. botulinum strain Hall A-hyper/tox⁻. Culture aliquots of a representative clone of Hall A-hyper/tox⁻/rBoNT/A4 and Hall A-hyper/tox⁻/rBoNT/A4-His were collected at indicated time points during growth, and both reduced (R) and nonreduced (NR) samples of each aliquot were analyzed on a 4 to 12% Bis-Tris gel with MOPS. Total culture lysates and purified botulinum neurotoxin isolated from strain Hall A-hyper were used as standards, and 120-h cultures of the expression host strain with and without the expression vector (no recombinant gene insert) were used as negative controls. For toxin detection, polyclonal antibody raised against BoNT/A1 (A and B) and monoclonal antibody against the His tag (C) were used.

FIG 3

BoNT/A1 and rBoNT/A4 complexes and toxins. Samples of purified BoNT/A1 and rBoNT/A4 before and after separation of the complex proteins were analyzed on a 4 to 12% Bis-Tris gel and Coomassie stained. The holotoxin, BoNT, reduced HC and LC, and complex proteins HA50, HA33, HA20, HA17, and NTNH are indicated. Lane 9, molecular mass markers; lane 1, A1 complex Hall A-hyper strain, unreduced (NR); lane 2, A1 complex Hall A-hyper, reduced (R); lane 3, rBoNT/A4 complex, unreduced; lane 4, rBoNT/A4 complex, reduced; lane 5, purified BoNT/A1; lane 6, purified BoNT/A1, reduced; lane 7, purified rBoNT/A4, unreduced; lane 8, purified rBoNT/A4, reduced.

FIG 4

Toxicity of rBoNT/A4 and BoNT/A4-L260F I264R mutant. (A) The in vitro catalytic activity of rBoNT/A4 and rBoNT/A4-L260F I264R was determined in a 2-h BoTest in relation to BoNT/A1. (B) Activity in human neurons was determined by an NCB assay. Neurons derived from hiPSCs were exposed to toxin dilutions for 48 h, and cell lysates were analyzed by Western blotting for SNAP-25 cleavage. Uncleaved SNAP-25 and cleaved SNAP-25 were quantified by densitometry, and the averages and standard deviations of triplicate samples were graphed using PRISM6 software.