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Dissecting and targeting ubiquitin networks in the course of bacterial infections

Periodic Reporting for period 4 - Ub-BAC (Dissecting and targeting ubiquitin networks in the course of bacterial infections)

Reporting period: 2022-01-01 to 2022-06-30

In the course of infection, bacterial pathogens inject hundreds of effector proteins into human cells to propagate their own proliferation and counteract the host’s defense mechanisms. Amongst the cellular pathways hijacked by bacteria is the ubiquitination system which regulates numerous essential processes within cells. The attachment of the small protein ubiquitin to other proteins changes their 3D structure and function, enabling new interactions amongst proteins and a fine-tuned regulation of signaling. Ubiquitination impacts on almost all cellular functions in higher organisms, including the defense mechanisms against pathogens. Whilst microbial pathogens lack this system, facultative intracellular bacteria like Salmonella, Shigella and Legionella have evolved multiple strategies to manipulate the host ubiquitin system to their own benefit. Analyzing the fundamental mechanisms of this hostile takeover helps us to better understand the course of bacterial infections and is key to the development of novel antibiotic strategies. With multi-resistant bacterial pathogens causing an increasing global health burden, this research is very relevant to society as it may reveal new druggable targets in the enduring fight against infections.
The central objective of the project is to analyze and understand the dynamic changes forced upon a host cell’s proteins in the course of a bacterial invasion with Salmonella, Shigella or Legionella. By this approach, novel pathways triggered by bacterial effector enzymes are identified and their contribution to pathogenicity and virulence characterized, as well as their suitability for being targeted in a therapeutic setting. The comprehensive datasets generated will constitute an invaluable resource which will be freely available to the entire scientific community.
A cornerstone to the infection efficiency of Salmonella is its ability to manipulate host defense mechanisms. To better understand the interaction between Salmonella and host cells, we set out to identify key host proteins that are modified by ubiquitin or phosphate groups and are involved in fighting off Salmonella. Therefore, the signature of all modifications was analyzed by state-of-the-art mass spectrometry before and after a Salmonella infection. This provided a global understanding of changes induced by the pathogen and a deep insight into how the two different modifications influence and depend on each other. We found that upon infection, both modifications interact in a deeply interconnected and highly dynamic way, targeting central infection-associated cellular signalling pathways related to regulation of inflammation, cell growth as well as death, trafficking, transport and autophagy. Our proteomic approaches in collaboration with Christian Münch’s lab (Hahn et al., PNAS 2021) revealed a time-resolved host translation response involving IKK/NF-κB activation and inhibition of the host integrated stress response (ISR) during Salmonella infection. In particular, we identified a regulatory link between the IKK kinase complex and the ISR (Hahn et al., EMBO reports, under revision).
Besides, our studies on intracellular pathways revealed that lipopolysaccharides (LPS) of Salmonella triggers interactions and clustering of ER membrane proteins including Fam134 family members. Multiple in vitro and in vivo studies indicate a direct interaction between LPS and FAM134B that mediates clustering of ER-phagy receptor complexes.
We further characterized the druggable proteome, especially proteins which had a relevant enzymatic activity (ligase, deubiquitinase or kinase) and were differentially regulated upon infection. Amongst those proteins was FAM49B/CYRI, for which we uncovered a hitherto unknown role in conferring resistance against Salmonella infection (Yuki et al., Nature Microbiology 2019).
For Shigella, we focused on a specific family of injected effectors known as “NEL-type bacterial ligases” which are capable of ubiquitinating cellular proteins and operate by a yet not understood mechanism. The identification of host proteins and cellular signaling pathways modified by this subfamily of bacterial effectors is essential to fully understand their contribution to virulence and pathogenicity and to exploit their significant potential as new drug targets. Again, we have performed a mass spectrometric analysis to characterize all ubiquitin modifications introduced by three members of this subfamily of bacterial effectors, and have provided a detailed ubiquitinome of bacterial ligases.
Lastly, we completed our investigation on the role of virulence effectors and host factors in response to infection by virulent Shigella flexneri in collaboration with Genentech (Maculins et al, eLife 2021). We identified multiple novel PTMs triggered by S. flexneri effectors that control components of inflammatory cytokine signaling, innate sensing and the core autophagy machinery. We continue dissecting the role of individual E3 ligases along these pathways. Furthermore, this study provided a comprehensive, multidimensional catalogue of proteome-wide changes in macrophages following infection by S. flexneri.
Upon infection, Legionella injects more than 300 effector proteins which take control over the host cell’s metabolism. Legionella has a unique system of modifying the cellular ubiquitin pool by phosphoribosylation, and we were at the forefront of laboratories dissecting this novel mechanism. This new type of ubiquitin (PR-Ubiquitin) is then transferred by Legionella SidE effector enzymes to host proteins by an unusual mechanism. By using biochemistry, structural biology and mass spectrometry, we were able to demonstrate the chemical basis of catalytic reaction mediated by the SidE-type ubiquitin ligases (Kalyil et al., Nature 2018). We also assessed the relevance of this modification during the course of infection and confirmed that the pathologic effect is exerted by ubiquitination of cellular target proteins. The discovery of this phosphoribose (PR)-dependent ubiquitination upon Legionella infection opened multiple avenues to explore the mechanistic details of this pathway. We have been pioneering many new concepts and were able to identify another Legionella effector – SidJ – which modifies SidE family of ubiquitin ligases by glutamylation and thereby inhibits their action (Bhogaraju et al., Nature 2019). Whilst the purpose of this is still unclear, it is tempting to speculate that the pathogen controls its own pathogenicity by this negative regulation loop. Recently, we identified DUPs as a new group of deubiquitinases from Legionella that can specifically reverse the action of SidE family members by removing the unusual PR-ubiquitin modification from target proteins (Shin et al., Molecular Cell 2020). DUPs have a specific role in control of organelle biogenesis and the infection cycles in host cells.
Lately, we identifyed and characterized novel substrates for the SidE ligase family in Legionella-infected cells. We were able to show that SidE effectors of Legionella modify the autophagic SNARE proteins STX17 and SNAP29 by serine ubiquitination. Time-resolved proximity labeling of STX17 revealed that in absence of LC3 conjugation, STX17 can still interact with other autophagy proteins like FIP200 and ATG13 in a serine ubiquitination dependent manner. We further discovered that bacterial vacuoles exhibit markers of both the endocytic and autophagic pathway and are formed by a fusion of Rab5 positive endosomes containing bacteria with STX17 positive pre-autophagosomes derived from the Golgi. The origin of these bacterial vacuoles may be similar to hybrid pre-autophagosomal structure (HyPAS) that has been recently characterized to be an important source of autophagic membranes (Mukherjee et al., Nature Microbiology under revision).
Taken together, by exploring ubiquitin and phosphorylation cascades during bacterial infections we have revealed multiple pathways with relevance for life cycle of bacteria and their pathogenesis.
3D structure of the enzymatic active part of SdeA toxin (taken from Kalayil et al., Nature 2018).