State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.
Front Cell Infect Microbiol. 2022 Apr 27;12:853204. doi: 10.3389/fcimb.2022.853204. eCollection 2022.
Cytolethal distending toxin (CDT) is a critical virulence factor of , and it induces cell death and regulates inflammation response in human epithelial cells. Pyroptosis is an inflammatory form of programmed cell death (PCD), but whether it is involved in CDT-mediated cytotoxicity remains elusive.
This study explores the role and mechanism of pyroptosis in CDT-mediated cytotoxicity.
HCT116 and FHC cell lines were treated with CDT. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability. Western blotting was used to measure the expression of related proteins in the pathway, and cell morphology observation, annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay were performed to evaluate the occurrence of pyroptosis.
Our results show that CDT effectively induces pyroptosis in a dose- and time- dependent manner in human colonic epithelial cells owing to its DNase activity. Specific pyroptotic features including large bubbles emerging from plasma membrane and LDH release were observed upon CDT treatment. Moreover, CDT-induced pyroptosis involves the caspase-9/caspase-3 axis, which is followed by gasdermin E (GSDME) cleavage rather than gasdermin D (GSDMD). N-acetyl cysteine (NAC), a reactive oxygen species (ROS) inhibitor, attenuates the activation of caspase-9/3, the cleavage of GSDME and pyroptotic characteristic, therefore demonstrating ROS initiates pyroptotic signaling.
We first clarify a molecular mechanism that CDT induces pyroptosis ROS/caspase-9/caspase-3/GSDME signaling. These findings provide a new insight on understanding of CDT-induced pathogenesis at the molecular level.
细胞致死膨胀毒素（CDT）是 的关键毒力因子，它在人类上皮细胞中诱导细胞死亡并调节炎症反应。细胞焦亡是一种程序性细胞死亡（PCD）的炎症形式，但它是否参与 CDT 介导的细胞毒性仍不清楚。
本研究探讨细胞焦亡在 CDT 介导的细胞毒性中的作用和机制。
用 CDT 处理 HCT116 和 FHC 细胞系。用细胞计数试剂盒-8（CCK-8）检测细胞活力。用 Western blot 检测通路中相关蛋白的表达，用细胞形态观察、膜联蛋白 V/碘化丙啶（PI）染色和乳酸脱氢酶（LDH）释放实验评估细胞焦亡的发生。
我们的结果表明，由于其 DNA 酶活性，CDT 能够有效地诱导人结肠上皮细胞中剂量和时间依赖性的细胞焦亡。在 CDT 处理后观察到质膜出现大泡和 LDH 释放等典型的细胞焦亡特征。此外，CDT 诱导的细胞焦亡涉及半胱氨酸天冬氨酸蛋白酶-9/半胱氨酸天冬氨酸蛋白酶-3 轴，随后是 GSDME 的切割而不是 GSDMD。活性氧（ROS）抑制剂 N-乙酰半胱氨酸（NAC）可减弱 caspase-9/3 的激活、GSDME 的切割和细胞焦亡特征，因此 ROS 可引发细胞焦亡信号。
我们首次阐明了 CDT 诱导细胞焦亡的分子机制，即 ROS/caspase-9/caspase-3/GSDME 信号通路。这些发现为理解 CDT 诱导的发病机制提供了新的认识。
