Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System
Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System
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DOI：
10.1128/aem.01795-14
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发表时间：
2014-12-01
影响因子：
4.4
通讯作者：
Johnson, Eric A.
中科院分区：
文献类型：
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作者：
Bradshaw, Marite;Tepp, William H.;Johnson, Eric A.
肉毒梭菌 A4 亚型神经毒素 (BoNT/A4) 在产生双毒素的肉毒梭菌菌株 657Ba 中自然表达，滴度比 BoNT/B 低 100 倍。在本研究中，我们描述了在非产孢且不产毒的肉毒杆菌表达宿主菌株中表达的重组 BoNT/A4 (rBoNT/A4) 的纯化。 rBoNT/A4 与表达宿主反式提供的无毒毒素复合物成分共纯化，并通过蛋白水解裂解为活性双链形式。重组BoNT/A4在小鼠和人神经元细胞中的活性比BoNT/A1低约1,000倍，并且重组BoNT/A4能被肉毒杆菌七价抗毒素有效中和。先前使用在大肠杆菌中表达的重组截短 BoNT/A4 轻链 (LC) 的报告表明，与 BoNT/A1 LC 相比，BoNT/A4 LC 的稳定性和活性降低，但通过引入单氨基酸取代 I264R 克服了这一问题。为了确定该突变是否也会影响 BoNT/A4 的全毒素活性，在梭菌表达系统中产生了携带该突变以及预计会增加溶解度的第二个突变 (L260F) 的重组全长 BoNT/A4。 rBoNT/A4 和 rBoNT/A4-L260F I264R 的体外、细胞和体内活性的比较分析显示，突变和未突变的 BoNT/A4 的活性均比 BoNT/A1 低 1,000 倍。这表明这些突变不会改变 BoNT/A4 全毒素的活性。总之，来自双毒素生产菌株的重组 BoNT 在内源梭菌表达系统中表达和纯化，从而可以分析该毒素。
Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100 x lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.