Lam Tina I, Stanker Larry H, Lee Kwangkook, Jin Rongsheng, Cheng Luisa W
Foodborne Toxin Detection and Prevention Unit, Western Regional Research Center, U.S. Department of Agriculture -Agricultural Research Service, Albany, CA, 94710, USA.
Physiology & Biophysics, School of Medicine, University of California, Irvine, CA, 92697, USA.
Cell Microbiol. 2015 Aug;17(8):1133-43. doi: 10.1111/cmi.12424. Epub 2015 Feb 15.
Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins. Botulinum neurotoxins associate with neurotoxin-associated proteins (NAPs) forming large complexes that are protected from the harsh environment of the gastrointestinal tract. However, it is still unclear how BoNT complexes as large as 900 kDa traverse the epithelial barrier and what role NAPs play in toxin translocation. In this study, we examined the transit of BoNT serotype A (BoNT/A) holotoxin, complex and recombinantly purified NAP complex through cultured and polarized Caco-2 cells and, for the first time, in the small mouse intestine. Botulinum neurotoxin serotype A and NAPs in the toxin complex were detectable inside intestinal cells beginning at 2 h post intoxication. Appearance of the BoNT/A holotoxin signal was slower, with detection starting at 4-6 h. This indicated that the holotoxin alone was sufficient for entry but the presence of NAPs enhanced the rate of entry. Botulinum neurotoxin serotype A detection peaked at approximately 6 and 8 h for complex and holotoxin, respectively, and thereafter began to disperse with some toxin remaining in the epithelia after 24 h. Purified HA complexes alone were also internalized and followed a similar time course to that of BoNT/A complex internalization. However, recombinant HA complexes did not enhance BoNT/A holotoxin entry in the absence of a physical link with BoNT/A. We propose a model for BoNT/A toxin complex translocation whereby toxin complex entry is facilitated by NAPs in a receptor-mediated mechanism. Understanding the intestinal uptake of BoNT complexes will aid the development of new measures to prevent or treat oral intoxications.
肉毒杆菌神经毒素（BoNTs）是一些毒性最强的天然毒素。肉毒杆菌神经毒素与神经毒素相关蛋白（NAPs）结合形成大的复合物，从而免受胃肠道恶劣环境的影响。然而，目前仍不清楚高达900 kDa的BoNT复合物如何穿过上皮屏障以及NAPs在毒素转运中发挥什么作用。在本研究中，我们检测了A型肉毒杆菌神经毒素（BoNT/A）全毒素、复合物和重组纯化的NAP复合物通过培养的极化Caco-2细胞的转运情况，并且首次在小鼠小肠中进行了检测。中毒后2小时开始在肠细胞内检测到毒素复合物中的A型肉毒杆菌神经毒素和NAPs。BoNT/A全毒素信号出现得较慢，4-6小时开始检测到。这表明单独的全毒素足以进入细胞，但NAPs的存在提高了进入速率。复合物和全毒素的A型肉毒杆菌神经毒素检测峰值分别约在6小时和8小时出现，此后开始分散，24小时后上皮细胞中仍残留一些毒素。单独的纯化HA复合物也被内化，并且其时间进程与BoNT/A复合物内化相似。然而，在与BoNT/A没有物理连接的情况下，重组HA复合物不会增强BoNT/A全毒素的进入。我们提出了一个BoNT/A毒素复合物转运模型，即毒素复合物的进入在受体介导的机制中由NAPs促进。了解BoNT复合物的肠道摄取将有助于开发预防或治疗经口中毒的新措施。
