Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA.
Department of Bacteriology, Kanazawa University, Kanazawa, Ishikawa, Japan.
mBio. 2024 Mar 13;15(3):e0310623. doi: 10.1128/mbio.03106-23. Epub 2024 Feb 13.
Botulinum neurotoxins (BoNTs) are a class of toxins produced by () and other species of . BoNT/X is a putative novel botulinum neurotoxin identified through genome sequencing and capable of SNARE cleavage, but its neurotoxic potential in humans and vertebrates remained unclear. The strain producing BoNT/X, Strain 111, encodes both a plasmid-borne as well as the chromosomal putative . This study utilized Strain 111 from Japan as well as recombinantly produced full-length BoNT/X to more fully analyze this putative pathogenic toxin. We confirmed production of full-length, catalytically active native BoNT/X by Strain 111, produced as a disulfide-bonded dichain polypeptide similar to other BoNTs. Both the purified native and the recombinant BoNT/X had high enzymatic activity but displayed very low potency in human-induced pluripotent stem cell-derived neuronal cells and in mice. Intraperitoneal injection of up to 50 µg of native BoNT/X in mice did not result in botulism; however, mild local paralysis was observed after injection of 2 μg into the gastrocnemius muscle. We further demonstrate that the lack of toxicity by BoNT/X is due to inefficient neuronal cell association and entry, which can be rescued by replacing the receptor binding domain of BoNT/X with that of BoNT/A. These data demonstrate that BoNT/X is not a potent vertebrate neurotoxin like the classical seven serotypes of BoNTs.
The family of botulinum neurotoxins comprises the most potent toxins known to humankind. New members of this family of protein toxins as well as more distantly related homologs are being identified. The discovery of BoNT/X via bioinformatic screen in 2017 as a putative new BoNT serotype raised concern about its potential as a pathogenic agent with no available countermeasures. This study for the first time assessed both recombinantly produced and native purified BoNT/X for its vertebrate neurotoxicity.
肉毒梭菌神经毒素（BoNTs）是一类由梭菌和其他种属产生的毒素。BoNT/X 是通过基因组测序鉴定的一种假定新型肉毒梭菌神经毒素，能够切割 SNARE，但它在人类和脊椎动物中的神经毒性潜力尚不清楚。产生 BoNT/X 的 111 株菌株，编码质粒细胞和染色体上假定的 BoNT/X。本研究利用来自日本的 111 株菌株以及重组产生的全长 BoNT/X 来更全面地分析这种假定的致病毒素。我们证实了 111 株菌株产生全长、具有催化活性的天然 BoNT/X，其产生方式类似于其他 BoNTs 的二硫键结合双链多肽。纯化的天然和重组 BoNT/X 均具有高酶活性，但在人诱导多能干细胞衍生的神经元细胞和小鼠中显示出非常低的效价。在小鼠中注射高达 50μg 的天然 BoNT/X 不会导致肉毒中毒；然而，在将 2μg 注射到腓肠肌后观察到轻微的局部瘫痪。我们进一步证明，BoNT/X 的缺乏毒性是由于其与神经元细胞的结合和进入效率低下，这可以通过用 BoNT/A 的受体结合结构域替换 BoNT/X 的受体结合结构域来挽救。这些数据表明，BoNT/X 不是像经典的七种 BoNTs 血清型那样的强效脊椎动物神经毒素。
肉毒梭菌神经毒素家族包含了人类已知的最有效毒素。这种蛋白质毒素家族的新成员以及更远缘的同源物正在被发现。2017 年通过生物信息学筛选发现 BoNT/X 作为一种假定的新型 BoNT 血清型引起了人们对其作为一种潜在致病剂的担忧，因为目前还没有可用的对策。本研究首次评估了重组产生的和天然纯化的 BoNT/X 的脊椎动物神经毒性。
