Exploring the Strategies of Bacterial Subversion of the Host Ubiquitin System: The Mechanism of Novel E3 Ligases (NEL) from Shigella

The aim of this project is to elucidate the mechanism by which bacterial ubiquitin ligases undermine the innate immune response of an infected cell. Our understanding will help to uncover new therapeutic strategies to fight the emerging threat of antimicrobial resistance. The innate immune response is the first line of defence against bacterial infections. It involves activation of a variety of cellular reactions, which cause inflammation of the infected tissue. The activation steps of the inflammatory response depend to a great extent on the ubiquitylation machinery of the cell. The ubiquitylation machinery consists of a cascade of different enzymes, which act in sequence to modify target proteins with ubiquitin chains. Ubiquitin chains consist of the small protein ubiquitin. The attachment of ubiquitin chains is tightly regulated and catalysed by a class of enzymes known as E3 ubiquitin ligases. Many pathogenic bacteria deploy a set of E3 ubiquitin ligases which they introduce into the cytosol of the infected host cell. These bacterial ubiquitin ligases, known as NELs (Novel E3 Ligase), are capable to modify components of the host ubiquitylation machinery with ubiquitin. The activity of the bacterial NELs interferes with normal function of the host ubiquitylation machinery which ultimately subverts the innate immune response in favour of the invading pathogen. It is currently unknown how NELs highjack the host ubiquitylation machinery to modify their target proteins. The human pathogen Shigella flexneri are highly infectious bacteria which are responsible for a severe form of dysentery known as shigellosis. Shigellosis a significant cause of morbidity and mortality worldwide. Some NELs from Shigella are targeting the linear ubiquitin chain assembly complex LUBAC which results in functional interference of the complex. LUBAC is a key component of the ubiquitylation machinery and required for the activation of the inflammatory response upon bacterial infection. We will use a combination of structural and biophysical techniques to determine the molecular mechanism by which the NEL IpaH1.4 from Shigella modifies LUBAC with ubiquitin and uncover how this modification inhibits LUBAC activity. Our findings will shed light on the molecular mechanism of Shigella pathogenesis and may provide new routes to restrict bacterial infections.

The NEL (novel E3 ligase) family of bacterial virulence factors constitutes a molecular weaponry of invading pathogens. Gram-negative bacteria deploy the Type 3 Secretion System to deliver effector proteins into the cytosol of the infected host cell. Effectors interact with host proteins to manipulate cellular processes and enable bacterial uptake. Innate immune signalling depends to a great extent on the modification of proteins with Ub chains. The activation requires the coordinated interplay of several E3 ubiquitin ligases. A class of effectors known as NELs exhibit ligase activity and exploit the host ubiquitylation cascade. The human pathogen Shigella flexneri utilizes NELs to establish itself in the host cell by targeting components of the ubiquitylation machinery. Substrates include the linear ubiquitin chain assembly complex LUBAC, an E3 enzyme which is required for the activation of the inflammatory response upon bacterial infection. Although the biochemical and cellular functions of bacterial effector proteins from Shigella are subjects of intensive studies, because they are likely to illuminate the molecular mechanisms of bacterial pathogenesis, the mechanism of IpaH ligases has not been determined. We will reconstitute the enzymatic cascade for ubiquitylation of the host protein LUBAC by the NEL IpaH1.4 from Shigella and elucidate the ubiquitin ligation mechanism in molecular detail. We will reach this goal by
1) using X-ray crystallography to determine an atomic model of IpaH1.4 in complex with its substrate LUBAC and ubiquitin, conjugated with an E2 enzyme
2) using fluorescence based techniques as well as AUC, ITC and SEC-MALS to establish a kinetic model of the ubiquitin ligation mechanism and elucidate how NEL ligase activity is regulated
3) use in vitro reconstitution assays and fluorescence based methods to uncover the molecular determinants for the functional restrictions of LUBAC caused by IpaH mediated Ub modification

Impact on public health
The research of this project is relevant to anyone who is interested in the development of new opportunities to tackle the growing problem of antibiotic resistance. Bacterial NEL E3s have the potential to become a target for new antimicrobial therapeutics. Hence, our finding will be of interest for the development of an NEL specific inhibitor. Our findings shall have a great impact to public health.

Impact on commercialisation
This project involves the further development of a new method based on a fluorescence based ligase activity assay. We anticipate that this assay is attractive for the biotech sector since it allows robust screening of ubiquitin ligase activity in a high throughput format which is useful for drug screening of various E3 ubiquitin ligases. We expect that the R&D sections of biopharma industries will be interested to apply our technology.

Impact on public awareness
Antimicrobial resistance is an emerging threat on a global scale. The research of this project might provide new therapeutic strategies for shigellosis. Dissemination of our findings outside the academic sector will increase the public awareness about the growing problem of drug resistance and increase the social acceptance of publicly funded research in the area of host-pathogen interactions.

Impact on knowledge transfer
The proposed research will facilitate the training of a postdoctoral research associate to gain highly advanced skills in biophysical and structural techniques, project management, scientific writing and presentation. This training will have a great impact on the development of his/her future career in academia, the biotech industry or the public sector. In addition, the knowledge gained from this project will be implemented in a taught module for undergraduate students.