The MOH Key Lab of Thrombosis and Hemostasis, The First Affiliated Hospital of Soochow University, Soochow University, Suzhou, China.
Department of Laboratory Examination, People's Hospital of Rizhao City, Rizhao, China.
Front Cell Infect Microbiol. 2021 Nov 2;11:724912. doi: 10.3389/fcimb.2021.724912. eCollection 2021.
We previously demonstrated the immunostimulatory efficacy of flagellar hook protein FlgE on epithelial cells, presumably ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE-ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both and . The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.
我们之前已经证明了鞭毛钩蛋白 FlgE 对上皮细胞的免疫刺激作用，这可能是由于细胞膜上存在异位 ATP 合酶或 ATP5B 亚基。在这里，我们使用重组野生型 FlgE、突变型 FlgE（FlgEM；在两个假定的关键表位 B 和 F 上发生突变）和 FlgE 类似物进行下拉实验、Western blot、流式细胞术和 ELISA，证实了 FlgE 蛋白或表位 B/F 肽与 ATP5B 的实际结合。在用 FlgE 蛋白处理后，人脐静脉内皮细胞（HUVEC）和 SV40 永生化的鼠血管内皮细胞表现出增殖、迁移、管形成和表面 ATP 产生减少，以及凋亡增加。FlgE 蛋白增加了 HUVEC 单层对可溶性大分子（如葡聚糖）以及中性粒细胞的通透性。免疫荧光显示 FlgE 诱导了 HUVEC 中 F-肌动蛋白的聚集和共轭。在携带移植实体瘤的 Balb/c-nude 小鼠中，FlgE 蛋白诱导了耳廓、肺部、肿瘤块和腹腔的微血管高通透性。FlgE 蛋白的所有作用在 FlgEM 蛋白中部分或完全受损，或在用抗 ATP5B 抗体预处理时被阻断。在细菌与 HUVEC 共培养时，FlgE 可在 HUVEC 的膜和细胞质中检测到。研究结论认为，FlgE 是异位 ATP5B 的致病配体，FlgE-ATP5B 偶联在上皮细胞上后，可调节内皮细胞的特性并增加其通透性。FlgE-异位 ATP5B 二聚体可能有助于与细菌感染或异位 ATP5B 阳性细胞相关的疾病的发病机制。
