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Dissecting and targeting ubiquitin networks in the course of bacterial infections

Periodic Reporting for period 2 - Ub-BAC (Dissecting and targeting ubiquitin networks in the course of bacterial infections)

Reporting period: 2019-01-01 to 2020-06-30

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. Ubiquitination involves the attachment of the small protein ubiquitin to other proteins, thereby changing their 3D structure and their function. 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 will help 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 of very high relevance 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.
All cellular functions are tightly controlled, and one mechanism to achieve this is by attaching small chemical molecules (e.g. phosphate groups) or even entire proteins (e.g. ubiquitin) to other proteins, thereby changing their 3D structure and their properties. These modifications enable new interactions amongst proteins and a fine-tuned regulation of cellular signaling. In the course of bacterial infections, the system is also hijacked by bacteria to their own advantage. In the first reporting period, we mainly focused on understanding modifications introduced and signalling pathways triggered by Salmonella, Shigella and Legionella infection.
A cornerstone to the infection efficiency of Salmonella is its ability to manipulate host defense mechanisms. In order 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. To achieve this, the signature of all modifications was analyzed by state-of-the-art mass spectrometry before and after a Salmonella infection. This enabled us to not only gain a global understanding of changes induced by the pathogen, but also provided 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 and cell growth as well as death, trafficking, transport and autophagy.
For further functional characterization we decided to focus on the druggable proteome, especially on proteins which had a relevant enzymatic activity (ligase, deubiquitinase or kinase) and were found as being differentially regulated upon infection. Amongst those proteins was FAM49B/CYRI, for which we were able to uncover 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.
A major part of our work during the first reporting period has concentrated on Legionella. Legionella causes Legionnaires’ disease characterized by an atypical form of pneumonia. 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 unusual 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 by this negative regulation loop controls its own pathogenicity. Most 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). We showed that DUPs have a specific role in control of organelle biogenesis and the infection cycles in host cells.
Taken together, by exploring ubiquitin and phosphorylation cascades during bacterial infections we have reveled multiple pathways with relevance for life cycle of bacteria and their pathogenesis.
In the next reporting period, we plan to further follow up on the molecular details of the Legionella enzyme families that control the life cycle of bacteria as well as cell metabolism of host cells and organelle changes. These appear to be critical for proliferation and maturation of pathogenic bacteria that are able to spread and damage the lung tissue. Moreover, by reaching a complete characterization of proteins that get modified by ubiquitin or phosphate groups after a Salmonella- or Shigella infection, we will be able to functionally characterize their roles. Even more ambitiously, we plan to use computation biology and multiple approaches using molecular dynamics and artificial intelligence to predict and monitor infection steps of Salmonella and Shigella.
3D structure of the enzymatic active part of SdeA toxin (taken from Kalayil et al., Nature 2018).