Isolation and Characterization of the Novel Botulinum Neurotoxin A Subtype 6

Abstract

Botulinum neurotoxins (BoNTs), the most potent toxins known to humans and the causative agent of botulism, exert their effect by entering motor neurons and cleaving and inactivating SNARE proteins, which are essential for neurotransmitter release. BoNTs are proven, valuable pharmaceuticals used to treat more than 200 neuronal disorders. BoNTs comprise 7 serotypes and more than 40 isoforms (subtypes). BoNT/A1 is the only A-subtype used clinically due to its high potency and long duration of action. While other BoNT/A subtypes have been purified and described, only BoNT/A2 is being investigated as an alternative to BoNT/A1. Here we describe subtype BoNT/A6 with improved pharmacological properties compared to BoNT/A1. It was isolated from Clostridium botulinum CDC41370, which produces both BoNT/B2 and BoNT/A6. The gene encoding BoNT/B2 was genetically inactivated, and A6 was isolated to greater than 95% purity. A6 was highly potent in cultured primary rodent neuronal cultures and in human induced pluripotent stem cell-derived neurons, requiring 20-fold less toxin to cause 50% SNAP-25 cleavage than A1. Second, A6 entered hiPSCs faster and more efficiently than A1 and yet had a long duration of action similar to BoNT/A1. Third, BoNT/A6 had similar LD₅₀ as BoNT/A1 after intraperitoneal injection in mice; however, local intramuscular injection resulted in less systemic toxicity than BoNT/A1 and a higher (i.m.) LD₅₀, indicating its potential as a safer pharmaceutical. These data suggest novel characteristics of BoNT/A6 and its potential as an improved pharmaceutical due to more efficient neuronal cell entry, greater ability to remain localized at the injection site, and a long duration.IMPORTANCE Botulinum neurotoxins (BoNTs) have proved to be an effective treatment for a large number of neuropathic conditions. BoNTs comprise a large family of zinc metalloproteases, but BoNT/A1 is used nearly exclusively for pharmaceutical purposes. The genetic inactivation of a second BoNT gene in the native strain enabled expression and isolation of a single BoNT/A6 from cultures. Its characterization indicated that BoNT/A subtype A6 has a long duration of action comparable to A1, while it enters neurons faster and more efficiently and remains more localized after intramuscular injection. These characteristics of BoNT/A6 are of interest for potential use of BoNT/A6 as a novel BoNT-based therapeutic that is effective and has a fast onset, an improved safety profile, and a long duration of action. Use of BoNT/A6 as a pharmaceutical also has the potential to reveal novel treatment motifs compared to currently used treatments.

Keywords: BoNT; BoNT/A6; botulinum neurotoxin; cell entry; duration; potency; subtype.

FIG 1

Isolation of BoNT/A6. The Clostridium botulinum strain CDC41370 was genetically modified to no longer produce BoNT/B2 as described in Materials and Methods. The purity of BoNT/A6 was verified by this SDS-PAGE gel showing the nonreduced (NR) holotoxin and reduced (R) heavy chain (HC) and light chain (LC).

FIG 2

LC activity of BoNT/A1 and /A6 after 2 h. Serial dilutions of BoNT/A1 and /A6 were analyzed for light chain activity using a FRET endopeptidase assay (BoTest, Biosentinel). Proteolytic activity of BoNT/A in real time was detected using 3 independent experiments, and averages and standard deviations from the three independent assays are shown. The BoTest uses a truncated SNAP-25 reporter construct of amino acids 141 to 206 flanked by a cyan florescent protein (CFP) and a yellow fluorescent protein (YFP), which leads to fluorescence emissions reflecting cleavage of SNAP-25.

FIG 3

SNAP-25 cleavage of BoNT/A1 and /A6 in hiPSCs. hiPSCs were exposed to serial dilutions of either A1 or A6 for 48 h. Cell lysates were analyzed for cleaved and uncleaved SNAP-25 by Western blotting and densitometry. Averages and standard deviations of triplicate samples are shown. The EC₅₀s were determined in PRISM6 software using a nonlinear regression (four parameters).

FIG 4

Cell entry kinetics of BoNT/A6 compared to /A1 and /A2. hiPSC-derived neurons were exposed to 70 pM BoNT/A1, /A2, or /A6 for up to 10 h, and cell lysates were prepared and analyzed for SNAP-25 cleavage at the indicated time points.

FIG 5

EC₅₀ and duration of action of BoNT/A6 and /A1 in neurons. Human iPSC-derived neurons were exposed to serial dilutions of BoNT/A6 (A) or /A1 (B) for 72 h, followed by BoNT removal. Cells were incubated further in toxin-free medium and harvested on days 3, 39, and 70 after exposure. Graphs generated in PRISM6 software depicting the average and standard deviation of triplicate samples are shown (A and B). The EC₅₀ values were determined in PRISM6 using a nonlinear regression (four parameters). The half-lives of BoNT/A1 and /A6 in hiPSCs were estimated by plotting the EC₅₀ values versus time and using the formula t_1/2 = ln(2)/slope of regression line (C). The half-lives were similar, being ∼12 and ∼14 days for A1 and A6, respectively. The duration of action was also evaluated in RSCs (D). RSCs were exposed to 8 pM BoNT/A6 for 3 days, followed by complete toxin removal. Cells were incubated for a longer duration in toxin-free medium, and cell lysates were prepared and analyzed for cleaved/uncleaved SNAP-25 by Western blotting and densitometry monthly until 8 months post-toxin exposure. Averages and standard deviations of quadruplicate samples are shown.

FIG 6

Onset and duration of action of BoNT/A6 and /A1 in vivo. Average DAS scores (A) and Rotarod times (B) of mice injected in the right gastrocnemius muscle with 0.6, 0.4, and 0.2 U of BoNT/A1 and BoNT/A6. n = 5.