Nonaka Takashi, Kuwabara Taku, Mimuro Hitomi, Kuwae Asaomi, Imajoh-Ohmi Shinobu
Division of Cell Biology and Biochemistry, Department of Basic Medical Sciences, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Division of Bacterial Infection, Department of Microbiology and Immunology, The Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Microbiology (Reading). 2003 Sep;149(Pt 9):2513-2527. doi: 10.1099/mic.0.26341-0.
It is currently unclear whether Shigella kills its phagocytic host cells by apoptosis or necrosis. This study shows that rapid necrosis ensues in macrophage-like cell lines (U937 cells differentiated by all-trans-retinoic acid and J774 cells) infected with the Shigella flexneri strain YSH6000. The infected cells rapidly lose membrane integrity, a typical feature of necrosis, as indicated by the release of the cytoplasmic lactate dehydrogenase and the exposure of phosphatidylserine (PS) associated with the rapid uptake of propidium iodide (PI). The infected cells exhibit DNA fragmentation without nuclear condensation, and substantial involvement of either caspase-3/-7 or caspase-1 was not detected, which is also contrary to what is normally observed in apoptosis. Cytochalasin D potently inhibited Shigella-induced cell death, indicating that only internalized Shigella can cause necrosis. Osmoprotectants such as polyethylene glycols could suppress cell death, suggesting that insertion of a pore by Shigella into the host cell membrane induces the necrosis. The pore was estimated to be 2.87+/-0.4 nm in diameter. Shigella was also found to be able to induce apoptosis but only in one of the lines tested and under specific conditions, namely U937 cells differentiated with interferon-gamma (U937IFN). Caspase-3/-7 but not caspase-1 activation was observed in these infected cells and the exposure of PS occurred without the uptake of PI. An avirulent Shigella strain, wild-type Shigella killed with gentamicin, and even Escherichia coli strain JM109, could also induce apoptosis in U937IFN cells, and cytochalasin D could not prevent apoptosis. It appears therefore that Shigella-induced apoptosis of U937IFN cells is unrelated to Shigella pathogenicity and does not require bacterial internalization. Thus, Shigella can induce rapid necrosis of macrophage-like cells in a virulence-related manner by forming pores in the host cell membrane while some cells can be killed through apoptosis in a virulence-independent fashion.
目前尚不清楚志贺氏菌是通过凋亡还是坏死杀死其吞噬性宿主细胞。本研究表明，感染弗氏志贺氏菌菌株YSH6000的巨噬细胞样细胞系（经全反式维甲酸分化的U937细胞和J774细胞）会迅速发生坏死。受感染的细胞迅速丧失膜完整性，这是坏死的典型特征，表现为细胞质乳酸脱氢酶的释放以及与碘化丙啶（PI）快速摄取相关的磷脂酰丝氨酸（PS）的暴露。受感染的细胞呈现DNA片段化但无核浓缩，且未检测到半胱天冬酶-3/-7或半胱天冬酶-1的大量参与，这也与凋亡中通常观察到的情况相反。细胞松弛素D能有效抑制志贺氏菌诱导的细胞死亡，表明只有内化的志贺氏菌才能导致坏死。聚乙二醇等渗透保护剂可抑制细胞死亡，提示志贺氏菌在宿主细胞膜上形成孔道会诱导坏死。估计该孔道直径为2.87±0.4纳米。还发现志贺氏菌能够诱导凋亡，但仅在一种测试细胞系中且在特定条件下，即经干扰素-γ分化的U937细胞（U937IFN）中。在这些受感染的细胞中观察到半胱天冬酶-3/-7而非半胱天冬酶-1的激活，且PS的暴露发生在未摄取PI的情况下。无毒力的志贺氏菌菌株、用庆大霉素杀死的野生型志贺氏菌，甚至大肠杆菌菌株JM109，也能在U937IFN细胞中诱导凋亡，且细胞松弛素D无法阻止凋亡。因此，志贺氏菌诱导U937IFN细胞凋亡似乎与志贺氏菌致病性无关，且不需要细菌内化。因此，志贺氏菌可通过在宿主细胞膜上形成孔道以与毒力相关的方式诱导巨噬细胞样细胞迅速坏死，而一些细胞可通过与毒力无关的方式通过凋亡被杀死。
