Xenophagy and bacterial avoidance

Final Report Summary - XABA (Xenophagy and bacterial avoidance)

Autophagy is an essential cellular process that enables cells to engulf and digest portions of their cytoplasm in a regulated manner, thereby accomplishing quality and quantity control of organelles, proteins and pathogens. These homeostatic and adaptive functions link intricately autophagy to diverse health and disease states including suppression of tumor development, prevention of neurodegeneration, and innate immunity.
Bacterial pathogens have evolved strategies to circumvent host cell defense mechanisms and promote their intercellular proliferation. Being an important part of the cellular immune response, the ubiquitin (Ub) system is a prominent target hijacked by bacterial effectors that are secreted to the host cytosol. Ubiquitination serves as a signal triggering NFKB activation, which by inducing inflammation dampens bacterial proliferation. Ub also marks bacteria for autophagosome-mediated degradation. While the majority of intracellular Salmonellae reside in the Salmonella-containing vacuole, a fraction of bacteria escapes to the cytosol where they are tagged with a dense polyubiquitin coat. Ub-binding autophagy cargo receptors subsequently mediate delivery of ubiquitinated bacteria to phagophores via interactions with human ATG8 proteins. Despite the key role of Ub during bacterial infection, the extent of host ubiquitinome remodeling induced by pathogen insults remained unexplored. Another unanswered question concerns the identity and origin of the ubiquitination targets constituting the Ub coat. Using complementary quantitative proteomics and image-based screening approaches we i) revealed ubiquitination-mediated regulation of Rho GTPase and NF-kB activities, mapped substrates of K48 and linear polyubiquitination, and determined host targets of bacterial E3 ligases/DUBs, ii) demonstrated extensive ubiquitination of bacterial effectors and outer membrane proteins, iii) identified the Ub ligase ARIH1 as one of the ligases involved in the formation of ubiquitin coat on cytosolic S. Typhimurium and iv) showed that together with LRSAM1 and HOIP, ARIH1 forms a network of E3 ligases that recognize cytosolic bacteria and mediate xenophagic degradation and host immune response.
Once engulfed in autophagosomes, bacteria and other intracellular material are delivered to lysosomes for degradation. This process requires Rab GTPases for proper membrane trafficking, since they control vesicle budding, targeting and fusion. However, their regulation especially through the activity of Rab GAPs and GEFs remains largely elusive. By employing complementary genetics and proteomics approaches we identified i) LC3- and GABARAP-interacting Rab GAPs as important regulators of autophagosome formation and maturation, ii) a GAP and GEF pair that control the recycling pool of ATG9 required for initiation of autophagy, iii) SMCR8 as multifaceted negative autophagy regulator.
While canonical autophagy, i.e. lysosomal degradation of cytosolic content, requires all of the core autophagy-related proteins, we revealed the role of autophagy proteins in pathways far removed from the canonical autophagy pathway (e.g. spatial substrate ubiquitylation, maintenance of ER exit sites and non-autophagic viral replication). This expansion of our understanding of noncanonical pathways, and the growing list of these pathways, supports the need to better clarify the context in which these pathways initiate, operate, and function.

Final Report Summary - XABA (Xenophagy and bacterial avoidance)

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