Functional dissection of ARIH1 E3 ubiquitin ligase in cellular antimicrobial immunity

肠沙门氏菌（S. enterica）是一种活动的革兰氏阴性细菌，是世界范围内食物或水传播感染的主要原因之一。感染肠炎沙门氏菌血清型鼠伤寒沙门氏菌（S. Typhimurium）导致肠胃炎与致命的结果免疫功能低下的病人。入侵鼠伤寒沙门氏菌的泛素化引发胞质细菌的自噬并限制其在上皮细胞中的扩散。泛素（Ub）链招募自噬受体，启动双膜自噬体结构的形成和溶酶体的破坏，这一过程被称为异噬。我们最近发现环间（RBR） Ub E3连接酶ARIH1（也称为hari）是宿主细胞泛素化机制中针对胞质沙门氏菌的重要新组成部分。有趣的是，由ARIH1贡献的Ub片段似乎具有抗菌功能，而不仅仅是作为异食性进食信号。尽管取得了这些进展，我们仍然缺乏对ARIH1和ARIH1依赖性泛素化在沙门氏菌感染应答中的作用的机制见解。为了缩小我们对这一重要宿主-病原体相互作用的理解上的这一主要差距，我们将把Alpi和Behrends实验室在ARIH1生物化学和宿主-病原体蛋白质组学方面的高度互补的专业知识与一系列微生物学工具和细胞生物学方法结合起来。通过将组织培养细胞中的沙门氏菌感染研究与广泛的结构引导的ARIH1相关试剂、体外检测、高分辨率共聚焦和活细胞成像以及接近和相互作用蛋白质组学相结合，我们的目标是确定：i) ARIH1是如何招募到胞质细菌的？ii)如何在沙门氏菌中激活E3连接酶ARIH1 ？iii) arih1产生的Ub片段对沙门氏菌的功能贡献是什么？我们的合作方法有可能揭示i)新的病原体相关分子模式（PAMPs），它触发细菌募集和激活ARIH1和ii) ARIH1或其相互作用蛋白之一作为各自的模式识别受体（PRRs）。加深我们对沙门氏菌ARIH1介导的泛素化机制的理解是一个有价值的目标，因为它将为开发新的分子工具来操纵ARIH1对其他细菌病原体的活性提供基础，从而为推动宿主-病原体相互作用的研究提供机会。

Salmonella enterica (S. enterica) is a motile Gram-negative bacterium that represents one of the major causes of food- or water-borne infections worldwide. Infection with S. enterica serovar Typhimurium (S. Typhimurium) results in gastroenteritis with fatal outcome in immuno-compromised patients. Ubiquitination of invading S. Typhimurium triggers autophagy of cytosolic bacteria and restricts their spread in epithelial cells. Ubiquitin (Ub) chains recruit autophagy receptors, which initiate the formation of double-membrane autophagosomal structures and lysosomal destruction in a process known as xenophagy. We recently identified the Ring-between-Ring (RBR) Ub E3 ligase ARIH1 (also known as HHARI) as an important novel component of the host cell ubiquitination machinery targeting cytosolic Salmonella. Intriguingly, Ub moieties contributed by ARIH1 seems to have anti-bacterial functions beyond their role as xenophagic eat-me signal. Despite these advances, we still lack mechanistic insights into the role of ARIH1 and ARIH1-dependent ubiquitination in response to Salmonella infection. To close this major gap in our understanding of this important host-pathogen interaction, we will combine the highly complementary expertise in ARIH1 biochemistry and host-pathogen proteomics of the Alpi and Behrends lab with a range of microbiology tools and cell biology approaches. By pairing Salmonella infection studies in tissue culture cells with an extensive panel of structure-guided ARIH1-related reagents, in vitro assays, high-resolution confocal and live-cell imaging as well as proximity and interaction proteomics, we aim at determining: i) How is ARIH1 recruited to cytosolic bacteria? ii) How is the E3 ligase ARIH1 activated at Salmonella? iii) What is the functional contribution of the ARIH1-generated Ub moieties on Salmonella? Our collaborative approach has the potential to uncover i) novel pathogen-associated molecular patterns (PAMPs) which triggers bacterial recruitment and activation of ARIH1 and ii) ARIH1 or one of its interacting proteins as their respective pattern recognition receptors (PRRs). Deepen our mechanistic understanding of ARIH1-mediated ubiquitination of Salmonella is a worthwhile goal as it will provide the basis to develop novel molecular tools to manipulate the activity of ARIH1 towards other bacterial pathogens, thereby providing an opportunity to drive research on host-pathogen interactions into new directions.