Functional dissection of ARIH1 E3 ubiquitin ligase in cellular antimicrobial immunity

肠道沙门氏菌（S.肠杆菌）是一种能动的革兰氏阴性细菌，其代表全世界食源性或水源性感染的主要原因之一。链球菌感染肠道血清型鼠伤寒沙门氏菌（S.鼠伤寒沙门氏菌）导致胃肠炎，在免疫功能低下的患者中具有致死性结局。入侵S.鼠伤寒沙门氏菌触发胞质细菌的自噬并限制它们在上皮细胞中的扩散。泛素（Ub）链募集自噬受体，启动双膜自噬体结构的形成和溶酶体破坏的过程称为异嗜。我们最近确定了环间环（RBR）Ub E3连接酶ARIH 1（也称为HHARI）作为靶向胞质沙门氏菌的宿主细胞泛素化机制的重要新组分。有趣的是，由ARIH 1贡献的Ub部分似乎具有抗菌功能，超出了它们作为异嗜性eat-me信号的作用。尽管取得了这些进展，我们仍然缺乏对ARIH 1和ARIH 1依赖性泛素化在沙门氏菌感染中的作用的机制见解。为了缩小我们对这一重要的宿主-病原体相互作用的理解方面的这一重大差距，我们将联合收割机将Alpi和Behrds实验室在ARIH 1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相结合。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ARIH 1相关试剂、体外测定、高分辨率共聚焦和活细胞成像以及邻近和相互作用蛋白质组学配对，我们旨在确定：i）ARIH 1如何被招募到胞质细菌中？ii）E3连接酶ARIH 1在沙门氏菌中是如何被激活的？iii）ARIH 1产生的Ub部分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）新型病原体相关分子模式（PAMP），其触发细菌招募和ARIH 1的激活，以及ii）ARIH 1或其相互作用蛋白之一作为其各自的模式识别受体（PRR）。加深我们对ARIH 1介导的沙门氏菌泛素化的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提供基础，以操纵ARIH 1对其他细菌病原体的活性，从而为将宿主-病原体相互作用的研究推向新的方向提供机会。

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.