Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College, London United Kingdom, London, United Kingdom.
Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research, London, United Kingdom.
mBio. 2018 Apr 10;9(2):e00170-18. doi: 10.1128/mBio.00170-18.
Tyrosine phosphorylation is key for signal transduction from exogenous stimuli, including the defense against pathogens. Conversely, pathogens can subvert protein phosphorylation to control host immune responses and facilitate invasion and dissemination. The bacterial effectors EspJ and SeoC are injected into host cells through a type III secretion system by enteropathogenic and enterohemorrhagic (EPEC and EHEC, respectively), , and , where they inhibit Src kinase by coupled amidation and ADP-ribosylation. , which is used to model EPEC and EHEC infections in humans, is a mouse pathogen triggering colonic crypt hyperplasia (CCH) and colitis. Enumeration of bacterial shedding and CCH confirmed that EspJ affects neither tolerance nor resistance to infection. However, comparison of the proteomes of intestinal epithelial cells isolated from mice infected with wild-type or encoding catalytically inactive EspJ revealed that EspJ-induced ADP-ribosylation regulates multiple nonreceptor tyrosine kinases Investigation of the substrate repertoire of EspJ revealed that in HeLa and A549 cells, Src and Csk were significantly targeted; in polarized Caco2 cells, EspJ targeted Src and Csk and the Src family kinase (SFK) Yes1, while in differentiated Thp1 cells, EspJ modified Csk, the SFKs Hck and Lyn, the Tec family kinases Tec and Btk, and the adapter tyrosine kinase Syk. Furthermore, Abl (HeLa and Caco2) and Lyn (Caco2) were enriched specifically in the EspJ-containing samples. Biochemical assays revealed that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, controls immune responses and the Src/Csk signaling axis. Enteropathogenic and enterohemorrhagic (EPEC and EHEC, respectively) strains cause significant mortality and morbidity worldwide. is a mouse pathogen used to model EPEC and EHEC pathogenesis Diarrheal disease is triggered following injection of bacterial effectors, via a type III secretion system (T3SS), into intestinal epithelial cells (IECs). While insights into the role of the effectors were historically obtained from pathological, immunologic, or cell culture phenotypes, subtle roles of individual effectors are often masked. The aim of this study was to elucidate the role and specificity of the ADP-ribosyltransferase effector EspJ. For the first time, we show that the processes affected by a T3SS effector can be studied by comparing the proteomes of IECs extracted from mice infected with wild-type or an catalytic mutant. We show that EspJ, the only bacterial ADP-ribosyltransferase that targets mammalian kinases, regulates the host immune response .
酪氨酸磷酸化是外源性刺激信号转导的关键，包括抵御病原体。相反，病原体可以颠覆蛋白质磷酸化来控制宿主免疫反应，并促进入侵和传播。细菌效应物 EspJ 和 SeoC 通过肠致病性和肠出血性大肠杆菌（EPEC 和 EHEC）的 III 型分泌系统注入宿主细胞，其中它们通过偶联酰胺化和 ADP-核糖基化抑制Src 激酶。使用该模型可模拟人类的 EPEC 和 EHEC 感染，是一种引发结肠隐窝增生（CCH）和结肠炎的小鼠病原体。细菌脱落和 CCH 的计数证实 EspJ 既不影响感染的耐受性也不影响感染的抵抗力。然而，比较野生型或编码无催化活性 EspJ 的感染小鼠的肠上皮细胞的蛋白质组表明，EspJ 诱导的 ADP-核糖基化调节多种非受体酪氨酸激酶。对 EspJ 的底物谱的研究表明，在 HeLa 和 A549 细胞中，Src 和 Csk 是显著的靶点；在极化的 Caco2 细胞中，EspJ 靶向 Src 和 Csk 以及Src 家族激酶（SFK）Yes1，而在分化的 Thp1 细胞中，EspJ 修饰 Csk、SFKs Hck 和 Lyn、 Tec 家族激酶 Tec 和 Btk 以及衔接酪氨酸激酶 Syk。此外，Abl（HeLa 和 Caco2）和 Lyn（Caco2）在 EspJ 存在的样品中特异性富集。生化测定表明，EspJ 是唯一靶向哺乳动物激酶的细菌 ADP-核糖基转移酶，可控制免疫反应和 Src/Csk 信号轴。肠致病性和肠出血性大肠杆菌（EPEC 和 EHEC）菌株在全球范围内造成严重的死亡率和发病率。是一种用于模拟 EPEC 和 EHEC 发病机制的小鼠病原体。腹泻病是通过细菌效应物通过 III 型分泌系统（T3SS）注入肠上皮细胞（IECs）引起的。虽然历史上对效应物作用的了解是通过病理学、免疫学或细胞培养表型获得的，但个别效应物的微妙作用往往被掩盖。本研究旨在阐明 ADP-核糖基转移酶效应物 EspJ 的作用和特异性。我们首次表明，通过比较野生型或催化突变体感染小鼠的 IECs 提取的蛋白质组，可以研究 T3SS 效应物影响的过程。我们表明，EspJ 是唯一靶向哺乳动物激酶的细菌 ADP-核糖基转移酶，可调节宿主免疫反应。
