2型猪链球菌膜囊泡通过激活NLRP3/Caspase-1/GSDMD信号通路诱导内皮细胞焦亡

doi:10.1016/j.jia.2023.09.022

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (4): 1338-1353.DOI: 10.1016/j.jia.2023.09.022

2型猪链球菌膜囊泡通过激活NLRP3/Caspase-1/GSDMD信号通路诱导内皮细胞焦亡

收稿日期:2023-04-16 接受日期:2023-11-09 出版日期:2024-04-20 发布日期:2024-03-30

Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Keda Shi^{1, 2, 3*}, Yan Li^{1, 2*}, Minsheng Xu^{1, 2, 3}, Kunli Zhang^{1, 2}, Hongchao Gou^{1, 2}, Chunling Li^{1, 2#},Shaolun Zhai^{1, 2#}

¹Institute of Animal Health, Guangdong Academy of Agricultural Sciences/Key Laboratory of Livestock Disease Prevention of Guangdong Province/Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, China
²Maoming Branch, Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Maoming 525000, China
³College of Animal Science & Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China

Received:2023-04-16 Accepted:2023-11-09 Online:2024-04-20 Published:2024-03-30
About author:Keda Shi, E-mail: Kedashi1994@163.com; Yan Li, E-mail: cyhlly@126.com; #Correspondence Chunling Li, E-mail: lclclare@163.com; Shaolun Zhai, E-mail: zhaishaolun@163.com * These authors contributed equally to this study.
Supported by:
This project was supported by the National Natural Science Foundation of China (U22A20520), the Innovation Team Project of Modern Agricultural Industrial Technology System of Guangdong Province, China (2023KJ119) and the Natural Science Foundation Program of Guangdong Province, China (2023A1515012206).

摘要/Abstract

摘要：

膜囊泡（membrane vesicles，MVs）可以主动分泌到细菌细胞外环境中，参与细菌和宿主的相互作用。大多数革兰氏阴性菌可以通过释放脂多糖(lipopolysaccharide，LPS)结合的外膜囊泡（out membrane vesicles，OMV），激活Caspase-11介导的非经典炎症通路，引起宿主细胞发生焦亡。然而，革兰氏阳性菌2型猪链球菌（Streptococcus suis serotype 2，S. suis 2）分泌的MVs引起细胞发生焦亡的机制尚不清楚。本研究利用超高速离心法、密度梯度离心、电子显微镜分析、蛋白质组学质谱鉴定、SDS-PAGE蛋白电泳和蛋白免疫印迹方法，分离、鉴定和表征MVs；利用荧光探针示踪分析、蛋白质印迹、荧光定量PCR和生物测定等方法，探究S. suis 2分泌的MVs与宿主的相互关系。研究结果显示，分离并鉴定S. suis 2来源的MVs大小范围在50 nm到400 nm之间，平均直径为72.04 nm，具有膜结构。蛋白质组学质谱鉴定表明，MVs携带200种细菌蛋白质，其中包含6个已知的S. suis 2的毒力蛋白。MVs主要通过动力蛋白依赖的内吞作用被转运到内皮细胞中，并激活内皮细胞中的NLRP3/Caspase-1/GSDMD经典炎症小体信号通路，产生活化的Caspase-1并切割GSDMD，形成焦亡的执行者—GSDMD-N，诱导细胞发生焦亡，向细胞外释放活化的IL-1β和LDH，但MVs不激活内皮细胞的Caspase-4/-5非经典通路。此外，内皮细胞在S. suis 2来源的MVs的诱导下产生大量活性氧（ROS）并导致线粒体失去膜电位引起线粒体DNA（mtDNA）向胞质释放，胞质中ROS和mtDNA的累积可能是激活NLRP3炎性小体的原因。本研究揭示了S. suis 2分泌的MVs通过动力蛋白依赖性内吞作用被内皮细胞内化，并可能通过损伤细胞线粒体诱导mtDNA、ROS的产生和释放，从而激活细胞NLRP3/Caspase-1/GSDMD经典通路，诱导内皮细胞发生焦亡。本研究首次揭示了S. suis 2通过分泌MVs激活内皮细胞NLRP3/Caspase-1/GSDMD经典通路，诱导内皮细胞发生焦亡的作用机制。

Abstract: Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that clinically causes severe swine and human infections (such as meningitis, endocarditis, and septicemia). In order to cause widespread diseases in different organs, S. suis 2 must colonize the host, break the blood barrier, and cause exaggerated inflammation. In the last few years, most studies have focused on a single virulence factor and its influences on the host. Membrane vesicles (MVs) can be actively secreted into the extracellular environment contributing to bacteria-host interactions. Gram-negative bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide (LPS), causing host cell pyroptosis. However, little is known about the effect of the MVs from S. suis 2 (Gram-positive bacteria without LPS) on cell pyroptosis. Thus, we investigated the molecular mechanism by which S. suis 2 MVs participate in endothelial cell pyroptosis. In this study, we used proteomics, electron scanning microscopy, fluorescence microscope, Western blotting, and bioassays, to investigate the MVs secreted by S. suis 2. First, we demonstrated that S. suis 2 secreted MVs with an average diameter of 72.04 nm, and 200 proteins in MVs were identified. Then, we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis. The S. suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway, resulting in cell pyroptosis, but it did not activate the Caspase-4/-5 pathway. More importantly, endothelial cells produce large amounts of reactive oxygen species (ROS) and lost their mitochondrial membrane potential under induction by S. suis 2 MVs. The results in this study suggest for the first time that MVs from S. suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage, which produced mtDNA and ROS under induction, leading to the pyroptosis of endothelial cells.

Key words: Streptococcus suis serotype 2 , membrane vesicles , endocytosis , pyroptosis , NLRP3 inflammasomes , mitochondrial damage , endothelial cell

. 2型猪链球菌膜囊泡通过激活NLRP3/Caspase-1/GSDMD信号通路诱导内皮细胞焦亡[J]. Journal of Integrative Agriculture, 2024, 23(4): 1338-1353.

Keda Shi, Yan Li, Minsheng Xu, Kunli Zhang, Hongchao Gou, Chunling Li, Shaolun Zhai.

Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway [J]. Journal of Integrative Agriculture, 2024, 23(4): 1338-1353.

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2型猪链球菌膜囊泡通过激活NLRP3/Caspase-1/GSDMD信号通路诱导内皮细胞焦亡

Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

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