Posttranslational Regulation of Botulinum Neurotoxin Production in Clostridium botulinum Hall A-hyper.
Posttranslational Regulation of Botulinum Neurotoxin Production in Clostridium botulinum Hall A-hyper.
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DOI：
10.1128/msphere.00328-21
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发表时间：
2021-08-25
期刊：
影响因子：
4.8
通讯作者：
Pellett S
中科院分区：
文献类型：
--
作者：
Inzalaco HN;Tepp WH;Fredrick C;Bradshaw M;Johnson EA;Pellett S
肉毒杆菌神经毒素(BoNTs)是人类已知的毒性最强的物质，也是神经麻痹疾病肉毒杆菌中毒的病原体。尽管BoNTs在公共卫生和安全方面具有重要意义，但作为一个生物恐怖主义问题，以及在药物开发中，人们对影响BoNTs在各种环境中稳定和降解的分子机制知之甚少。之前使用肉毒梭菌ATCC 3502菌株进行的研究表明，高水平的精氨酸(20g/升)可以抑制大约1000倍的BONT产量。在本研究中，我们利用Clostron技术对肉毒杆菌HALL A-HYPER菌株的精氨酸强化培养物中毒素的降低机制进行了探索。培养物生长在毒素产生培养基(TPM)和富含精氨酸的TPM中。分析培养物的生长情况(600 nm处的光密度[OD600])、pH值的变化以及骨形成和稳定性。我们的数据表明，肉毒杆菌菌株Hall A-超级培养过程中精氨酸的富集会导致pH漂移，从而引发依赖于pH的翻译后调控机制。我们进一步证明，在肉毒杆菌HALL A-HYPER的生长过程中，维持酸性培养物的pH不依赖于精氨酸，对毒素的稳定性起着核心作用，并且培养物产生的胞外金属蛋白水解酶在pH值为~6.5到8.0的范围内导致BONT的降解。重要性肉毒杆菌神经毒素(BoNT)是一种重要的、应用广泛的药物，也是一种重要的公共卫生和生物恐怖主义问题，但目前对其合成的调控还不是很清楚。本文强调了环境控制的翻译后调控机制对肉毒杆菌产生的生物活性BoNTs的加工和稳定性的影响。这项工作的结果将有助于加强公共卫生和安全措施，以及我们评估新型BoNTs安全风险的能力，并提高用于制药的BoNTs的产量和质量。
Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind and are the causative agents of the neuroparalytic disease botulism. Despite the overall importance of BoNTs in public health and safety, as a bioterrorism concern, and in pharmaceutical development, little is known about the molecular mechanisms mediating BoNT stability and degradation in various environments. Previous studies using Clostridium botulinum strain ATCC 3502 revealed that high levels of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold. In the present study, the mechanisms of toxin reduction in arginine-enriched cultures of C. botulinum strain Hall A-hyper, which we have previously genetically manipulated using ClosTron technology, were explored. Cultures were grown in toxin production medium (TPM) and TPM enriched with arginine. Cultures were analyzed for growth (optical density at 600 nm [OD600]), changes in pH, and BoNT formation and stability. Our data indicate that arginine enrichment of C. botulinum strain Hall A-hyper cultures results in a pH shift that induces pH-dependent posttranslational control mechanisms. We further show that independent of arginine, maintenance of an acidic culture pH during growth of C. botulinum strain Hall A-hyper plays a central role in toxin stability and that an extracellular metalloprotease produced by the culture results in BoNT degradation at pH levels between ⁓6.5 and 8.0. IMPORTANCE Botulinum neurotoxin (BoNT) is a public health and bioterrorism concern as well as an important and widely used pharmaceutical, yet the regulation of its synthesis by BoNT-producing clostridia is not well understood. This paper highlights the role of environmentally controlled posttranslational regulatory mechanisms influencing processing and stability of biologically active BoNTs produced by C. botulinum. The results of this work will help enhance public health and safety measures and our ability to evaluate safety risks of novel BoNTs and improve production and quality of BoNTs for pharmaceutical use.