Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.
Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.
复制标题
DOI：
10.1128/msphere.00100-15
复制
发表时间：
2016-01
期刊：
影响因子：
4.8
通讯作者：
Johnson EA
中科院分区：
文献类型：
--
作者：
Pellett S;Tepp WH;Bradshaw M;Kalb SR;Dykes JK;Lin G;Nawrocki EM;Pier CL;Barr JR;Maslanka SE;Johnson EA
由厌氧菌产生的肉毒神经毒素（BoNT）是潜在致命的神经麻痹性疾病肉毒中毒的原因。基于BoNT被同源抗血清选择性中和，BoNT被分为七种血清型，血清型A至G，这与临床和诊断目的相关。尽管支持性治疗显著降低了肉毒杆菌中毒的死亡率，但减少症状严重程度和恢复时间的唯一药物干预是早期给予抗毒素（针对BoNT产生的抗血清）。最近一份关于新的BoNT血清型（血清型H）的报告引起了人们对“治疗抗性”和高效毒素的关注。然而，毒素的嵌合结构和特征表明嵌合BoNT/FA。在这里，我们描述了这种新型毒素的纯化形式的第一个特征。BoNT/FA被可用的抗毒素中和，支持分类为BoNT/FA。BoNT/FA需要蛋白水解活化以实现完全毒性，并且与BoNT/A1相比在小鼠中具有相对较低的效力，但在培养的神经元中具有令人惊讶的高活性。肉毒杆菌神经毒素（BoNT）是由神经毒素梭菌产生的，是人类和动物严重疾病肉毒中毒的原因。对BoNT的早期研究已经基于其在小鼠中的毒性被同源抗体选择性中和而将其分类为七种血清型（血清型A至G）。最近，一份关于BoNT潜在的第八种血清型（命名为“H型”）的报告引起了争议。这种新的BoNT与BoNT/B2一起在产生双重毒素的肉毒梭菌菌株中产生。用于将这种新型毒素指定为新血清型的数据来自含有BoNT/B2和新型毒素的培养物上清液以及序列信息，尽管来自两个独立实验室的数据表明被针对BoNT/A1产生的抗体中和，并提出分类为BoNT/FA。序列数据表明嵌合结构由BoNT/A1受体结合结构域、BoNT/F5轻链结构域和与BoNT/F1最密切相关的新型易位结构域组成。在这里，我们描述了从天然菌株中纯化的这种毒素的表征，其中第二BoNT（BoNT/B）的表达已被消除。质谱分析表明，毒素制剂仅含有BoNT/FA，并证实了与BoNT/F5类似的催化活性。小鼠体内生物测定表明，该毒素的比活性为3.8 × 107小鼠50%致死剂量（mLD 50）单位/mg，而在培养的人神经元中的活性非常高（50%有效浓度[EC 50] = 0.02 mLD 50/孔）。细胞和小鼠中的中和试验均表明，针对BoNT/A1产生的各种抗体完全中和，尽管效率比BoNT/A1低16至20倍。重要性肉毒杆菌神经毒素（BoNT），由厌氧菌产生，是潜在致命的，神经麻痹性疾病肉毒中毒的原因。基于BoNT被同源抗血清选择性中和，BoNT被分为七种血清型，血清型A至G，这与临床和诊断目的相关。尽管支持性治疗显著降低了肉毒杆菌中毒的死亡率，但减少症状严重程度和恢复时间的唯一药物干预是早期给予抗毒素（针对BoNT产生的抗血清）。最近一份关于新的BoNT血清型（血清型H）的报告引起了人们对“治疗抗性”和高效毒素的关注。然而，毒素的嵌合结构和特征表明嵌合BoNT/FA。在这里，我们描述了这种新型毒素的纯化形式的第一个特征。BoNT/FA被可用的抗毒素中和，支持分类为BoNT/FA。BoNT/FA需要蛋白水解活化以实现完全毒性，并且与BoNT/A1相比在小鼠中具有相对较低的效力，但在培养的神经元中具有令人惊讶的高活性。
Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons. Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.