Departments of Genetics and Medicine, Stanford University School of Medicine, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2013 Oct 15;110(42):E4007-15. doi: 10.1073/pnas.1316852110. Epub 2013 Oct 1.
The protective antigen component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8, and β1-integrin, and, with the assistance of other host proteins, enters targeted cells by receptor-mediated endocytosis. Using an antisense-based phenotypic screen, we discovered the role of calpains in this process. We show that functions of a ubiquitous Ca(2+)-dependent cysteine protease, calpain-2, and of the calpain substrate talin-1 are exploited for association of anthrax toxin and its principal receptor, CMG2, with higher-order actin filaments and consequently for toxin entry into host cells. Down-regulated expression of calpain-2 or talin-1, or pharmacological interference with calpain action, did not affect toxin binding but reduced endocytosis and increased the survival of cells exposed to anthrax lethal toxin. Adventitious expression of wild-type talin-1 promoted toxin endocytosis and lethality, whereas expression of a talin-1 mutant (L432G) that is insensitive to calpain cleavage did not. Disruption of talin-1, which links integrin-containing focal adhesion complexes to the actin cytoskeleton, facilitated association of toxin bound to its principal cell-surface receptor, CMG2, with higher-order actin filaments undergoing dynamic disassembly and reassembly during endocytosis. Our results reveal a mechanism by which a bacterial toxin uses constitutively occurring calpain-mediated cytoskeletal rearrangement for internalization.
炭疽杆菌毒素的保护性抗原组分可以与宿主细胞表面上至少三种不同的蛋白质相互作用，即毛细血管形态发生基因 2（CMG2）、肿瘤内皮标记物 8 和β1-整合素，并在其他宿主蛋白的协助下通过受体介导的内吞作用进入靶细胞。我们使用反义基于表型筛选的方法发现钙蛋白酶在这个过程中的作用。我们表明，普遍存在的 Ca(2+)-依赖性半胱氨酸蛋白酶钙蛋白酶-2 的功能以及钙蛋白酶底物桩蛋白-1 的功能被用于炭疽毒素及其主要受体 CMG2 与高级肌动蛋白丝的关联，从而使毒素进入宿主细胞。钙蛋白酶-2 或桩蛋白-1 的下调表达，或用药理学方法干扰钙蛋白酶的作用，不会影响毒素的结合，但会减少内吞作用并增加暴露于炭疽致死毒素的细胞的存活率。野生型桩蛋白-1 的偶然表达促进了毒素的内吞作用和致死性，而对钙蛋白酶切割不敏感的桩蛋白-1 突变体（L432G）的表达则没有。桩蛋白-1 的破坏，将整合素包含的粘着斑复合物与肌动蛋白细胞骨架连接起来，促进了毒素与主要细胞表面受体 CMG2 结合，与在内吞作用过程中进行动态解聚和重组的高级肌动蛋白丝相关联。我们的结果揭示了一种细菌毒素利用组成性发生的钙蛋白酶介导的细胞骨架重排进行内化的机制。
