Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America.
Department of Veterinary Pathology, Iowa State University, Ames, Iowa, United States of America.
PLoS Pathog. 2024 May 17;20(5):e1011835. doi: 10.1371/journal.ppat.1011835. eCollection 2024 May.
A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes.
一类新型的杀菌化合物是苏云金芽孢杆菌（Bt）产生的 Crystal 3D（Cry）和细胞溶解（Cyt）蛋白。一些 Bt Cry 蛋白具有选择性杀线虫活性，其中 Cry5B 研究最多。Cry5B 通过选择性结合细胞膜糖脂，然后在肠细胞膜上形成孔，导致细胞损伤，从而杀死线虫寄生虫。Cry5B 选择性靶向来自不同进化枝的多种线虫物种，对哺乳动物宿主没有影响。左旋咪唑是一种胆碱能驱虫药，通过选择性打开在线虫肌肉上发现的 L 型烟碱型乙酰胆碱受体离子通道（L-AChRs）起作用。左旋咪唑和 Cry5B 在整个蠕虫水平上的协同杀线虫相互作用以前已有描述，但这种协同作用的位置、机制和时程尚不清楚。在这项研究中，我们使用荧光成像技术，跟踪左旋咪唑和 Cry5B 对线虫肠道肠细胞内 Ca2+水平的影响时间线。观察到左旋咪唑的峰值 Ca2+反应大约在 10 分钟后，而激活的 Cry5B 的峰值反应大约在 80 分钟后。当左旋咪唑和 Cry5B 同时应用时，我们观察到 Cry5B 的反应更大，发生得更早，而不是单独应用时。据推测，协同作用是由于左旋咪唑打开 Ca2+通透性 L 型烟碱型乙酰胆碱受体和在肠细胞质膜中产生的 Ca2+通透性 Cry5B 毒素孔引起的细胞质 Ca2+超载所致。左旋咪唑的作用增强并加速了 Cry5B 的作用，导致更大的 Ca2+超载，加速肠细胞死亡。
