A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin

Abstract

Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated as BoNT/H. The BoNT gene (bont) sequence of BoNT/H was compared to known bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to the structures of BoNT/A1 and BoNT/F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxins, including those contained in multivalent therapeutic antitoxin products that are the mainstay of human botulism treatment.

Keywords: Clostridium botulinum; botulinum toxin; botulism; neuronal cell-based assay; neutralization; serotype.

Figure 1.

A and C, Nucleotide similarity plots (derived from SimPlot [7]) are shown for the novel bont/FA (A) and the previously recognized bont/CD (C). The percentage similarity was generated using a 200-bp window and a 20-bp step. A, bont/A1 (green) and bont/F5 (red) are shown with bont/FA (individual nucleotide data were obtained from GenBank accession numbers AM412317 [for bont/A1], GU213212 [for bont/F5], and JSCF00000000 [for bont/FA]). B, bont/D (green) and bont/C (red) are shown with bont/CD (individual nucleotide data were obtained from GenBank accession numbers JENR01000128 [for bont/D], AB200358 [for bont/C], and AB200360 [for bont/CD]). The gene regions encoding the 3 domains (light chain [LC], N-terminal heavy chain [HC_N], and C-terminal heavy chain [HC_C]) are indicated by dotted lines. B and D, The predicted amino acid identity of botulinum neurotoxin (BoNT) LC, HC_N, and HC_C domains are for the hybrid toxins BoNT F/A (B) and BoNT C/D (D). Domains sharing ≥80% amino acid identity in pairwise alignments between the associated hybrid toxins and the comparison toxins are shaded. The percentage amino acid identity of the most similar domains is also indicated. The structure of the novel toxin contained in strain IBCA10-7060 (BoNT F/A) has significant similarity to the LC domain of BoNT/F5 (A) and the HC_C domain of BoNT/A1 (B). For comparison, the structure of another hybrid toxin (BoNT C/D) is also shown (C and D).

Figure 2.

Neutralization of University of Wisconsin–Madison (UW) toxin 2 with heptavalent botulism antitoxin (BAT) in a neuronal cell–based assay. The indicated amounts of extract were incubated without (top) or with 2 µL (bottom) of heptavalent botulism antitoxin, in 100 µL of culture medium and incubated for 1 hour at 37°C. The toxin/BAT mixtures were then added to hiPSC-derived neurons (100 µL/well) and incubated for 24 hours at 37°C in 5% CO₂. Cell lysates were prepared in 50 µL of lithium dodecyl sulfate sample buffer (Life Technologies) and analyzed by Western blot for levels of VAMP2, syntaxin, and SNAP-25, as previously described [24, 25]. VAMP-2 remains intact when the culture supernatant is pretreated with BAT, demonstrating a protective capacity.