Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.
Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury.
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DOI：
10.1371/journal.ppat.1006159
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发表时间：
2017-02
期刊：
影响因子：
6.7
通讯作者：
Karch H
中科院分区：
文献类型：
--
作者：
Bielaszewska M;Rüter C;Bauwens A;Greune L;Jarosch KA;Steil D;Zhang W;He X;Lloubes R;Fruth A;Kim KS;Schmidt MA;Dobrindt U;Mellmann A;Karch H
外膜囊泡（OMV）是细菌毒力的重要工具，但它们在肠出血性大肠杆菌（EHEC）O 157引起的感染的发病机制中的作用，危及生命的溶血性尿毒症综合征的主要原因，知之甚少。使用蛋白质组学，电子和共聚焦激光扫描显微镜，免疫印迹和生物测定，我们调查O 157肠出血性大肠杆菌临床分离株分泌的OMV的毒力因子货物，与致病相关的人类细胞的相互作用，和细胞损伤的机制。我们证明O 157 OMV携带EHEC O 157关键毒力因子的混合物，包括滋贺毒素2a（Stx 2a）、细胞致死膨胀毒素V（CdtV）、EHEC溶血素和鞭毛蛋白。毒素通过OMV的动力蛋白依赖性内吞作用被细胞内化，并在细胞内运输期间与囊泡差异分离。Stx 2a和CdtV-B（DNase样CdtV亚基）在早期内体中与OMV分离。Stx 2a与其受体神经酰胺globotriaosylceramide在抗洗涤剂膜内被运输到高尔基复合体和内质网，催化Stx 2a A1片段从内质网易位到胞质溶胶。CdtV-B在其逆行运输到内质网后，易位到细胞核以到达DNA。CdtV-A和CdtV-C亚基保持与OMV相关，并与OMV一起分选到溶酶体。EHEC溶血素在溶酶体中与OMV分离并靶向线粒体。OMV递送的CdtV-B引起细胞DNA损伤，其激活DNA损伤应答，导致G2细胞周期停滞。停滞的细胞最终死于Stx 2a和CdtV通过半胱天冬酶-9活化诱导的凋亡。通过证明天然分泌的EHEC O 157 OMV携带并向细胞中递送具有生物活性的毒力因子的混合物，从而导致细胞死亡，并通过对OMV和OMV递送的毒力因子的细胞内运输进行首次全面分析，我们对EHEC O 157感染的发病机制提供了新的见解。我们的数据对考虑O 157 OMV作为候选疫苗具有影响。肠出血性大肠杆菌（EHEC）O 157是引起人类腹泻和危及生命的溶血性尿毒综合征的主要EHEC群，它产生多种毒力因子，在这些疾病的发病机制中发挥不同的作用。然而，其分泌和宿主细胞损伤的机制知之甚少。我们发现从患者体内分离的肠出血性大肠杆菌O 157菌株具有称为外膜囊泡（OMV）的纳米结构，其中含有主要的肠出血性大肠杆菌O 157毒力因子，包括滋贺毒素2a（Stx 2a）、细胞致死膨胀毒素V（CdtV）、肠出血性大肠杆菌溶血素和鞭毛蛋白。OMV被人肠上皮细胞、肾和脑微血管内皮细胞摄取，这些细胞是EHEC O 157感染过程中的主要靶细胞，并在细胞内递送毒力因子。在细胞内，毒力因子与OMV分离，并通过不同的途径转运至其靶区室，包括细胞质（Stx 2a）、细胞核（CdtV-B亚基）和线粒体（EHEC溶血素）。暴露于EHEC O 157 OMV的细胞发生由CdtV-B介导的DNA损伤诱导的G2细胞周期阻滞。随后是由Stx 2a和CdtV通过半胱天冬酶-9激活触发的凋亡性细胞死亡。因此，OMV作为EHEC O 157介导的宿主损伤的新工具，很可能参与人类疾病的发病机制。
Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors, we provide new insights into the pathogenesis of EHEC O157 infections. Our data have implications for considering O157 OMVs as vaccine candidates. Enterohemorrhagic Escherichia coli (EHEC) O157, the leading EHEC group causing diarrhea and the life-threatening hemolytic uremic syndrome in humans, produce several virulence factors which play distinct roles in the pathogenesis of these diseases. However, the mechanisms of their secretion and host cell injury are poorly understood. We show here that EHEC O157 strains isolated from patients shed nanostructures termed outer membrane vesicles (OMVs) which contain major EHEC O157 virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The OMVs are taken up by human intestinal epithelial and renal and brain microvascular endothelial cells, which are the major targets during EHEC O157 infections, and deliver the virulence factors intracellularly. Inside cells the virulence factors separate from OMVs and are transported via different pathways to their target compartments including the cytosol (Stx2a), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). Cells exposed to EHEC O157 OMVs develop G2 cell cycle arrest induced by CdtV-B-mediated DNA damage. This is followed by apoptotic cell death triggered by Stx2a and CdtV via caspase-9 activation. OMVs thus serve as novel tools of EHEC O157-mediated host injury and are quite likely involved in the pathogenesis of human diseases.