Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

致病性梭菌会分泌强效毒素，导致宿主组织严重损伤。索氏拟梭菌致死毒素 (TcsL) 会引起与妇科感染相关的几乎总是致命的中毒性休克综合征。TcsL 与艰难梭菌TcdB有 87% 相似，后者通过结肠上皮中的卷曲受体进入宿主细胞。然而，P. sordellii感染的目标是血管内皮，这表明 TcsL 利用了另一种受体。在这里，我们使用 CRISPR/Cas9 筛选，建立了信号蛋白 SEMA6A 和 SEMA6B 作为 TcsL 受体。我们证明重组 SEMA6A 可以保护小鼠免受 TcsL 诱导的水肿。3.3 Å 冷冻电镜结构表明，TcsL 与 SEMA6A 的结合区域与 TcdB 中与结构无关的 Frizzled 的结合区域相同。值得注意的是，这个进化上不同的表面中的 15 个突变足以将 TcsL 的结合特异性转变为 TcdB 的结合特异性。我们的研究结果将信号蛋白确立为 TcsL 的生理相关受体，并揭示了高度相关毒素之间组织靶向和疾病发病机制差异的分子基础。