Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan.
Infect Immun. 2012 Aug;80(8):2886-93. doi: 10.1128/IAI.00302-12. Epub 2012 Jun 4.
Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.
肉毒梭菌 C 型和 D 型通过产生血清型特异性或镶嵌型肉毒神经毒素（BoNT）引起动物肉毒中毒。由日本牛源肉毒中毒分离株产生的 D/C 镶嵌型 BoNT（BoNT/DC）对小鼠的毒性最高。相比之下，大鼠对 C 型和 D 型 BoNT 及其镶嵌型 BoNT 中的 BoNT/DC 表现出很强的抗性。我们试图通过与 C 型和 D 型 BoNT（BoNT/C 和 BoNT/D）进行比较，来表征 BoNT/DC 的酶和受体结合活性。BoNT/DC 和 D 对来自小鼠的小脑颗粒细胞（CGC）表现出相似的毒性作用，但前者对大鼠 CGC 的毒性较小。在重组鼠源性囊泡相关膜蛋白（VAMP）中，两种 BoNT 对大鼠同工型 1 VAMP（VAMP1）的酶活性均低于对其他 VAMP 同源物的酶活性。然后，我们研究了神经节苷脂作为 C 型和 D 型以及镶嵌型 BoNT 结合成分的生理意义。发现 BoNT/DC 和 C 在外源性添加 GM1a 和 GT1b 神经节苷脂后分别能够切割 PC12 细胞的细胞内底物，表明每种 BoNT 识别不同的神经节苷脂部分。BoNT/DC 对 CGC 中谷氨酸释放的作用可被霍乱毒素 B 亚单位（CTB）阻断，但不能被不与 GM1a 结合的 CTB 定向突变体阻断。牛肾上腺嗜铬细胞对 BoNT/DC 的敏感性似乎高于 BoNT/C 和 D。这些结果表明，BoNT/DC 独特的受体结合机制可能在动物中差异调节其生物学活性。
