Functional dissection of ARIH1 E3 ubiquitin ligase in cellular antimicrobial immunity

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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.