Department of Molecular Genetics and Microbiology, and Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, United States of America.
PLoS One. 2012;7(4):e36019. doi: 10.1371/journal.pone.0036019. Epub 2012 Apr 26.
Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.
耶尔森氏外蛋白 J（YopJ）是一种 III 型分泌系统（T3SS）效应物，存在于致病性耶尔森氏菌（鼠疫耶尔森氏菌、小肠结肠炎耶尔森氏菌和假结核耶尔森氏菌）中，可被分泌到宿主细胞中。YopJ 抑制巨噬细胞中的存活反应途径，导致细胞死亡。YopJ 的等位基因变异导致耶尔森氏菌菌株的细胞毒性存在差异。小肠结肠炎耶尔森氏菌 O:8（YopP）和鼠疫耶尔森氏菌 KIM（YopJ（KIM））菌株中的 YopJ 同工型具有高细胞毒性活性。此外，YopJ（KIM）诱导的巨噬细胞死亡与半胱天冬酶-1 的激活和白细胞介素-1β（IL-1β）的分泌有关。在这里，研究了主要鼠巨噬细胞中 YopJ（KIM）诱导的细胞死亡、半胱天冬酶-1 激活和 IL-1β 分泌的机制。在鼠疫耶尔森氏菌 KIM5 感染的巨噬细胞中，半胱天冬酶-3/7 的活性较低，半胱天冬酶-3 底物多聚（ADP-核糖）聚合酶（PARP）未被切割。此外，在存在半胱天冬酶-8 抑制剂或 B 细胞淋巴瘤 2（Bcl-2）相关 X 蛋白（Bax）/Bcl-2 同源拮抗剂/杀伤（Bak）敲除巨噬细胞中，细胞毒性和 IL-1β 的分泌并没有减少，表明 YopJ（KIM）介导的细胞死亡和半胱天冬酶-1 的激活不依赖于线粒体导向的细胞凋亡。KIM5 感染的巨噬细胞释放高迁移率族蛋白 B1（HMGB1），这是一种坏死的标志物，显微镜分析显示，坏死细胞含有活性半胱天冬酶-1，表明半胱天冬酶-1 的激活与坏死有关。抑制剂研究表明，受体相互作用蛋白 1（RIP1）激酶和活性氧（ROS）不是 KIM5 感染巨噬细胞中细胞毒性或 IL-β 释放所必需的。在存在组织蛋白酶 B 抑制剂的情况下，IL-1β 的分泌减少，表明半胱天冬酶-1 的激活需要组织蛋白酶 B 的活性。与鼠疫耶尔森氏菌 KIM 相比，假结核耶尔森氏菌中过表达的 YopP 导致更高的细胞毒性和 IL-1β 的分泌。野生型和同基因半胱天冬酶 1 敲除 C57BL/6 小鼠对过表达 YopP 的假结核耶尔森氏菌的致死性感染具有相同的易感性。这些数据表明，YopJ 诱导的耶尔森氏菌感染巨噬细胞中的半胱天冬酶-1 激活是坏死细胞死亡的下游后果，对于具有增强的细胞毒性的菌株的固有宿主抗性是不必要的。
