Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand.
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phahonyothin Road, Khlong Luang, Phathum Thani 12120, Thailand.
J Invertebr Pathol. 2020 Mar;171:107340. doi: 10.1016/j.jip.2020.107340. Epub 2020 Feb 7.
Vip3Aa protein is produced by Bacillus thuringiensis during vegetative growth and displays high toxicity against a wide range of lepidopteran insect larvae such as Spodoptera exigua and Spodoptera frugiperda, both important insect pests worldwide. Vip3Aa protein is synthesized as a protoxin (proVip3Aa) and becomes activated by digestion with either trypsin or insect gut proteases. The activated Vip3Aa protein (actVip3Aa) binds to a specific receptor in the brush border epithelial midgut cells, causing cell death via apoptosis, possibly induced by its pore-forming activity. Here we investigated the actVip3Aa intracellular localization to explain the molecular mechanism leading to the cytotoxicity of Vip3Aa toxin. The Spodoptera frugiperda (Sf9) cell line was incubated with fluorescently labeled Vip3Aa, namely Alexa488-actVip3Aa, and the intracellular localization was analyzed through a laser scanning confocal microscope. The Alexa488-actVip3Aa was internalized into the Sf9 cells. Immunofluorescence detection demonstrated that Alexa488-actVip3Aa did not colocalize with early endosomes which is usually implicated in clathrin-mediated endocytosis, suggesting that the actVip3Aa does not use clathrin-dependent endocytosis to transport into the cytosol. Intracellular visualization also shows that actVip3Aa does not directly target to mitochondria upon entry into the cytosol. Following internalization, actVip3Aa causes cell division disruption that subsequently could trigger cell death via apoptosis.
Vip3Aa 蛋白是苏云金芽孢杆菌在营养生长阶段产生的，对鳞翅目昆虫如甜菜夜蛾和玉米螟等具有广谱毒性，这些昆虫都是全球重要的农业害虫。Vip3Aa 蛋白作为原毒素（proVip3Aa）合成，然后被胰蛋白酶或昆虫肠道蛋白酶消化而激活。激活的 Vip3Aa 蛋白（actVip3Aa）与肠道刷状缘上皮细胞中的特定受体结合，通过细胞凋亡导致细胞死亡，其可能是通过形成孔道的活性诱导细胞凋亡。在这里，我们研究了 actVip3Aa 的细胞内定位，以解释导致 Vip3Aa 毒素细胞毒性的分子机制。用荧光标记的 Vip3Aa（即 Alexa488-actVip3Aa）孵育美洲棉铃虫（ Sf9 ）细胞系，并通过激光扫描共聚焦显微镜分析细胞内定位。Alexa488-actVip3Aa 被内化到 Sf9 细胞中。免疫荧光检测表明，Alexa488-actVip3Aa 与早期内体不共定位，而早期内体通常与网格蛋白介导的内吞作用有关，这表明 actVip3Aa 不使用网格蛋白依赖性内吞作用进入细胞质。细胞内可视化还表明，actVip3Aa 进入细胞质后并不直接靶向线粒体。内化后，actVip3Aa 会导致细胞分裂紊乱，随后可能通过细胞凋亡引发细胞死亡。
