NleB host targets - The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD)

The inhibition of host innate immunity pathways is essential for the survival of attaching and effacing (A/E) pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during mammalian infections. To subvert these pathways, A/E pathogens utilize a type III secretion system (T3SS) to introduce effectors that target key signaling pathways thereby suppressing the anti-microbial response. One effector used by A/E pathogens is the arginine glycosyltransferase NleB1 (NleBCR in C. rodentium) that modifies conserved arginine residues with N-acetylglucosamine (GlcNAc) in death-domain containing host proteins thereby blocking extrinsic apoptosis signaling. When expressed ectopically, NleB1 modifies the host proteins, FADD, TRADD and RIPK1. However, the true repertoire of arginine-GlcNAcylation during infection with endogenous levels of NleB delivered by the pathogen is unknown. Here we explored the effects of arginine-GlcNAcylation by NleB on the global host proteome. Utilizing an affinity proteomic approach for Arginine-GlcNAcylated glycopeptide, we compared the global repertoire of arginine-GlcNAcylation during ectopic expression of NleB, EPEC infection in vitro or C. rodentium infection in vivo. When NleB was overexpressed, multiple host proteins were arginine-GlcNAcylated. However, when endogenous levels of NleB were delivered during EPEC and C. rodentium infection, R117of FADD was rapidly and preferentially modified. The arginine-GlcNAcylation modification of FADD was extremely stable and insensitive to environmental or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine-GlcNAcylation did not illicit any proteomic changes, even in response to prolonged expression of NleB. Thus, under wild type levels of expression, NleB1/NleBCR antagonizes death-receptor-induced apoptosis of infected cells by modifying FADD in an irreversible and silent manner.

宿主先天免疫通路的抑制对于黏附消除型（attaching and effacing, A/E）病原体——如肠致病性大肠杆菌（enteropathogenic Escherichia coli, EPEC）和鼠柠檬酸杆菌（Citrobacter rodentium）——在哺乳动物感染过程中的存活至关重要。为了颠覆此类免疫通路，A/E病原体借助III型分泌系统（type III secretion system, T3SS）将效应蛋白递送至宿主细胞，这些效应蛋白靶向关键信号通路，从而抑制抗菌免疫应答。A/E病原体所使用的一类效应蛋白为精氨酸糖基转移酶NleB1（在鼠柠檬酸杆菌中为NleBCR），其可在含死亡结构域的宿主蛋白的保守精氨酸残基上进行N-乙酰葡糖胺（N-acetylglucosamine, GlcNAc）修饰，进而阻断外源性凋亡信号通路。当异位表达NleB1时，该酶可修饰宿主蛋白FADD、TRADD与RIPK1。然而，在病原体以内源水平递送NleB的感染过程中，精氨酸-GlcNAcylation修饰的真实底物谱仍未明确。本研究探究了NleB介导的精氨酸-GlcNAcylation对宿主整体蛋白质组的影响。我们利用针对精氨酸-GlcNAcylated糖肽的亲和蛋白质组学方法，对比了NleB异位表达、体外EPEC感染或体内鼠柠檬酸杆菌感染过程中精氨酸-GlcNAcylation的整体底物谱。当NleB过表达时，有多款宿主蛋白发生了精氨酸-GlcNAcylation修饰。但当病原体在EPEC感染和鼠柠檬酸杆菌感染中以内源水平递送NleB时，FADD的R117位点会被快速且优先修饰。FADD的精氨酸-GlcNAcylation修饰极为稳定，且不受环境或宿主细胞降解机制的影响。尽管该修饰具有稳定性并能抑制凋亡，但精氨酸-GlcNAcylation并未引发任何蛋白质组学变化，即便在长期表达NleB的情况下亦是如此。综上，在野生型表达水平下，NleB1/NleBCR通过以不可逆且隐蔽的方式修饰FADD，从而拮抗死亡受体诱导的感染细胞凋亡。