RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin
RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin
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DOI：
10.1128/mbio.02985-19
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发表时间：
2019-11-01
期刊：
影响因子：
6.4
通讯作者：
McClane, Bruce A.
中科院分区：
文献类型：
--
作者：
Shrestha, Archana;Gohari, Iman Mehdizadeh;McClane, Bruce A.
F 型产气荚膜梭菌菌株产生一种名为产气荚膜梭菌肠毒素 (CPE) 的成孔毒素，会导致胃肠道疾病。在人肠细胞样 Caco-2 细胞中，低 CPE 浓度会导致 caspase-3 依赖性细胞凋亡，而高 CPE 浓度会导致坏死。由于坏死或细胞凋亡有时涉及受体相互作用的丝氨酸/苏氨酸蛋白激酶 1 或 3（RIP1 或 RIP3），因此本研究探讨了这些激酶是否对 CPE 诱导的细胞凋亡或坏死很重要。高度特异性的 RIP1 或 RIP3 抑制剂可减少 Caco-2 细胞中 CPE 诱导的细胞凋亡和坏死。这些发现表明，用高 CPE 浓度处理 Caco-2 细胞诱导的坏死形式涉及坏死性凋亡，当高而不是低 CPE 浓度显示诱导混合谱系激酶结构域样假激酶 (MLKL) 寡聚化（坏死性凋亡的关键后期步骤）时，这一点得到证实。此外，MLKL 寡聚化抑制剂减少了由高而不是低 CPE 浓度引起的细胞死亡。这些激酶的抑制剂支持 RIP1 和 RIP3 参与 CPE 诱导的坏死性凋亡，在高 CPE 浓度治疗期间也减少了 MLKL 寡聚化。钙蛋白酶抑制剂同样阻断高 CPE 浓度诱导的 MLKL 寡聚化，表明钙蛋白酶激活是启动 CPE 诱导的坏死性凋亡的关键中间体。在另外两种 CPE 敏感细胞系中，即 Vero 细胞和人肠细胞样 T84 细胞，低 CPE 浓度也主要引起凋亡/晚期凋亡，而高 CPE 浓度主要引起坏死性凋亡。总的来说，这些结果表明，高而不是低的 CPE 浓度会导致坏死性凋亡，并表明 RIP1、RIP3、MLKL 或钙蛋白酶抑制剂可以作为对抗体内 CPE 效应的潜在疗法。 重要性 产气荚膜梭菌 F 型菌株是食物中毒和抗生素相关性腹泻的常见原因。 F 型菌株毒力需要产生产气荚膜梭菌肠毒素 (CPE)。在 Caco-2 细胞中，高浓度的 CPE 会导致细胞坏死，而低浓度的肠毒素会诱导细胞凋亡。目前的研究确定，受体相互作用的丝氨酸/苏氨酸蛋白激酶1和3参与了Caco-2细胞中CPE诱导的细胞凋亡和坏死，而混合谱系激酶结构域样假激酶（MLKL）寡聚化参与了CPE诱导的坏死，从而表明这种形式的CPE诱导的细胞死亡涉及坏死性凋亡。高 CPE 浓度还会导致 T84 和 Vero 细胞坏死性凋亡。钙蛋白酶激活被认为是 CPE 诱导的坏死性凋亡的关键中间体。这些结果表明 RIP1、RIP3、MLKL 寡聚化或钙蛋白酶的抑制剂是对抗 CPE 介导的疾病的有用疗法。
Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivo.IMPORTANCE C. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.