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The Role of IFN-Γ in Cell-Autonomous Immune Responses Against Salmonella Typhimurium and Toxoplasma Gondii

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The Role of IFN-Γ in Cell-Autonomous Immune Responses Against Salmonella Typhimurium and Toxoplasma Gondii The role of IFN-γ in cell-autonomous immune responses against Salmonella Typhimurium and Toxoplasma gondii Solène C. Rolland Trinity College University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy March 2020 Summary Summary The research presented in this thesis investigates the function of interferon gamma in vacuoles, namely Salmonella enterica serovar Typhimurium and Toxoplasma gondii. IFN-γ is a cytokine that functions in both the innate and the adaptive immune responses. IFN-γ is mainly secreted by T cells and natural killer (NK) cells and activates macrophages, promotes antigen presentation and enhances antiviral and antibacterial immunity. In the case of bacterial pathogens, such as S.Typhimurium, priming with IFN-γ leads to an inflammatory but controlled cell death mechanism known as pyroptosis, following bacterial invasion of the cytosol or experimental lipopolysaccharide (LPS) transfection. LPS is a major component of the outer membrane of Gram-negative bacteria and is sensed in the cytosol by human Caspase-4. The research described in Chapter 2 used a genome-wide CRISPR-Cas9 screen as an unbiased approach to identify new IFN-γ-induced genes that play a role in the cell death pathway. This viability screen enriched for cells resistant to LPS-mediated cell death after IFN-γ priming. Their sequencing generated a list of potential candidate genes including expected genes such as IFN-γ receptors (Ifngr) and novel genes such as Nckap1, Fip1l1, Srsf10, Safb. However, none of these additional candidates were validated because their deficiency did not provide significant resistance to the cell death phenotype. Overall, these results suggest that, in addition to the Caspase-4- Gasdermin-D axis, the role of IFN-γ-induced genes in the cell death pathway triggered by cytosolic LPS remains to be investigated. This thesis also examined the role of IFN-γ in response to infection by the eukaryotic parasite Toxoplasma gondii, an apicomplexan that actively invades host cells and resides within a unique parasitophorous vacuole. The work presented in Chapter 3 showed that with type II T.gondii (PRU strain), IFN-γ inhibits replication in an autophagy-dependent manner. This strain is coated by ubiquitin and by the individual subunits (HOIP, HOIL-1, and Sharpin) of the linear-ubiquitin assembly complex (LUBAC). Autophagy cargo receptors, such as NDP52, p62, and Optineurin, are also recruited to the type II, intracellular parasites. Although all these recruitments were IFN-γ-dependent, the role of LUBAC and the mechanisms for its recruitment remain unknown. This research further showed that PRU changes cytokine expression levels of infected cells: IL-1α, IL-6, and CCL5 expressions are induced upon infection and potentially in a LUBAC-dependent fashion. Overall, the work presented in this thesis provides new insights into the role of IFN-γ in antibacterial and antiparasitic cell-autonomous innate immunity. Further experiments could validate other candidate genes from the screen or investigate the role of LUBAC in Toxoplasmosis. i Declaration Declaration This thesis is the result of my own work and includes nothing that is the outcome of work done in collaboration, except where specified in the text. The work in this thesis has not already been submitted before for any degree or for any other qualification. This thesis does not exceed the word limit of 60,000 words prescribed by the degree committee for the Faculty of Biology. Solene C. Rolland Trinity College University of Cambridge March 2020 ii Acknowledgements Acknowledgements I am grateful to my supervisor Felix Randow for allowing me to pursue my PhD under his guidance. I would also like to give thanks for the support I have received from past and current members of the Randow research group both for their help from a scientific perspective as well as for showing sympathy and sharing kind words when experiments were not working. My sincere gratitude goes to the following scientists for their particular impact on my PhD work: Jessica Noad, who trained me when I first joined the lab and provided numerous plasmids and some materials for my projects; Keith Boyle for his encouragement, advice, and honest criticisms on my thesis drafts; Michal Wandel for providing insights to my project and sharing his knowledge about IFN-γ; Claudio Pathe for helping me with microscopy-related experiments and for training me on the high-content microscope. Finally, I wish to thank Conor and Gisela for their kind attitude and for always being in a good mood, trying to cheer me up. I am grateful to the MRC and the LMB for funding this research and for providing a collaborative environment in which I could pursue and complete my PhD. I also wish to express my gratitude to the staff in the media kitchen for pouring numerous plates, and to the microscopy and flow cytometry teams for their training and maintenance of the microscopes and machines. I would also like to acknowledge the time that Clara Lloyd spent proof-reading and helping me format my thesis. She has been an important moral support throughout my PhD too. I am especially thankful to my family, who has been unwavering and constant in their support throughout my PhD. They kept me going and encouraged me during the difficult times. iii iv Table of contents Table of Contents Summary ................................................................................................................................. i Declaration ........................................................................................................................... ii Acknowledgements .......................................................................................................... iii Table of Contents ................................................................................................................ v Abbreviations ....................................................................................................................... x Chapter 1: Introduction .................................................................................................... 1 1.1 Immune defence mechanisms against pathogens .................................................... 1 1.1.1 Recognition mechanisms ......................................................................................................... 3 1.1.1.1. Pattern-recognition receptors surveil both extracellular and intracellular compartments ....................................................................................................................................................... 3 1.1.1.2. Inflammasomes are cell-autonomous sensing mechanisms ............................................ 3 1.1.1.3. Other mechanisms of intracellular detection .......................................................................... 7 1.1.2 Microbicidal pathways ............................................................................................................12 1.1.2.1. Autophagy .............................................................................................................................................12 1.1.2.2. Cell death ...............................................................................................................................................14 1.1.2.3. Phagocytosis ........................................................................................................................................19 1.1.3 Production and release of effector molecules makes up an integrated immune system .......................................................................................................................................................19 1.1.3.1. NF-κB signalling .................................................................................................................................20 1.1.3.2. Interferon signalling .........................................................................................................................23 1.1.3.3. Cytokine production .........................................................................................................................27 1.1.4 Immunity relies on multiple cross-talks to function as a fully integrated system .......................................................................................................................................................29 1.2 Two model organisms to study cell-autonomous immunity: Salmonella Typhimurium and Toxoplasma gondii ............................................................................... 30 1.2.1 Pathogenesis ...............................................................................................................................31 1.2.2 Entry and establishment of the vacuole ..........................................................................33 1.2.3 Host cell-autonomous immunity directed against S.Typhimurium and T.gondii ......................................................................................................................................................................38 1.2.4 Microbial effectors for a successful host invasion .......................................................45 Aims ...................................................................................................................................... 47 Chapter 2: A genome-wide screen to identify IFN-γ induced genes required for cell death in response to cytosolic LPS .............................................................
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