El Qaidi Samir, Chen Kangming, Halim Adnan, Siukstaite Lina, Rueter Christian, Hurtado-Guerrero Ramon, Clausen Henrik, Hardwidge Philip R
From the College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506.
the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
J Biol Chem. 2017 Jul 7;292(27):11423-11430. doi: 10.1074/jbc.M117.790675. Epub 2017 May 18.
Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with -acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic (EHEC), enteropathogenic (EPEC), and Moreover, strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg and Arg, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.
许多革兰氏阴性菌病原体利用一种名为III型分泌系统的注射器状装置将毒力因子注入宿主细胞。其中一些效应蛋白是酶，它们修饰宿主蛋白以颠覆其正常功能。NleB是一种糖基转移酶，它用N-乙酰-D-葡萄糖胺修饰宿主蛋白，以抑制宿主的抗菌和炎症反应。NleB在黏附/蚀损性病原体肠出血性大肠杆菌（EHEC）、肠致病性大肠杆菌（EPEC）中保守，此外，鼠柠檬酸杆菌菌株编码多达三个NleB直系同源物，分别命名为SseK1、SseK2和SseK3。然而，关于NleB/SseK直系同源物的活性和宿主蛋白靶点存在相互矛盾的报道。因此，我们在此进行了糖基化测定和细胞培养实验，以比较这些效应蛋白的活性和底物特异性。SseK1、SseK3、EHEC NleB1、EPEC NleB1和柠檬酸杆菌NleB阻断了肿瘤坏死因子（TNF）介导的核因子κB（NF-κB）信号通路激活，而SseK2和NleB2则没有。柠檬酸杆菌NleB、EHEC NleB1和SseK1使宿主甘油醛-3-磷酸脱氢酶（GAPDH）糖基化。柠檬酸杆菌NleB、EHEC NleB1、EPEC NleB1和SseK2使Fas相关死亡结构域蛋白（FADD）糖基化。SseK3和NleB2对这两种底物均无活性。我们还发现，EHEC NleB1使GAPDH的两个精氨酸残基（Arg171和Arg174）糖基化，并且这两个残基对于GAPDH介导的肿瘤坏死因子受体相关因子2（TRAF2）泛素化激活至关重要。这些结果提供了证据，表明这个高度保守的细菌毒力效应蛋白家族的成员靶向不同的宿主蛋白底物，并表现出不同的细胞作用模式来抑制宿主反应。
