Cytoskeletal rearrangement and acute cytotoxicity occur in Vibrio vulnificus-infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of V. vulnificus and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491-1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA11491-1971 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA11491-1971 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.

中文翻译：

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创伤弧菌RtxA1细胞毒素靶向纤维蛋白A，以调节PAK1和MAPK依赖的细胞骨架重组和细胞死亡。

在创伤弧菌感染的宿主细胞中发生细胞骨架重排和急性细胞毒性。RtxA1毒素是一种多功能的自动加工毒素重复序列（MARTX），对于V. vulnificus的发病机理和程序性坏死细胞死亡至关重要。在这项研究中，观察到表达与GFP融合的RtxA1氨基酸1491-1971的HeLa细胞是圆形的。通过酵母双杂交筛选和随后的免疫沉淀验证分析，我们证实了RtxA11491-1971片段与宿主细胞纤维蛋白A（肌动蛋白交联支架蛋白）的特异性结合。纤维蛋白A表达的下调降低了RtxA1对宿主细胞的细胞毒性。此外，在毒素介导的细胞死亡过程中，RtxA1-filamin A相互作用诱导了JNK和p38 MAPKs的磷酸化。然而，在缺乏纤维蛋白A的M2细胞的RtxA1中毒过程中未观察到这些MAPK的磷酸化。此外，pak1的耗竭似乎由RtxA1-filamin A相互作用激活，抑制了RtxA1诱导的JNK和p38磷酸化，用pak1抑制剂处理的细胞显示出RtxA1介导的细胞骨架重排和细胞毒性降低。因此，RtxA11491-1971结构域与纤维蛋白A的结合似乎是pak1介导的MAPK活化的必要条件，该活化导致细胞骨架重组和宿主细胞死亡。pak1抑制剂处理的细胞表现出减少的RtxA1介导的细胞骨架重排和细胞毒性。因此，RtxA11491-1971结构域与纤维蛋白A的结合似乎是pak1介导的MAPK活化的必要条件，该活化导致细胞骨架重组和宿主细胞死亡。pak1抑制剂处理的细胞表现出减少的RtxA1介导的细胞骨架重排和细胞毒性。因此，RtxA11491-1971结构域与纤维蛋白A的结合似乎是pak1介导的MAPK活化的必要条件，该活化导致细胞骨架重组和宿主细胞死亡。