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Toxoplasma gondii is an obligate intracellular parasite and paradigm for the study of the host immune response to infection at the cellular and molecular level. The zebrafish larva is a well-established model for the study of bacterial, viral and fungal infections in vivo. In this thesis, I use the zebrafish larva to set up a novel in vivo infection model for Toxoplasma. I use this model to explore innate leukocyte dynamics and function, and investigate the cell-intrinsic immune response to Toxoplasma infection. I also develop new tools for the in-depth study of cell-intrinsic mechanisms involved in parasite restriction. Recent studies have revealed fundamental differences in the innate host response to Toxoplasma infection between human and mice suggesting that another animal model could provide new insights into Toxoplasma control. In Chapter 1, I develop a zebrafish larval model to study Toxoplasma infection in vivo. Macrophages and neutrophils make up the cellular innate immune response, and are the first cells to respond to pathogen invasion. Studies using mice have shown that both macrophages and neutrophils are crucial for the in vivo control of Toxoplasma. However, different reports suggest that Toxoplasma can exploit macrophage and / or neutrophil biology to promote their survival. In Chapter 2, I use the Toxoplasma-zebrafish infection model developed in Chapter 1 to reveal that macrophages significantly contribute to Toxoplasma clearance in vivo. Moreover, I test for strain-specific differences in establishing infection, and show that type II and III strains establish a higher parasite burden than type I strains in vivo. Guanylate binding proteins (GBPs) are important mediators of IFN-γ-induced cell-intrinsic restriction of Toxoplasma. GBPs have been implicated in multiple cell-intrinsic pathways, including host cell death. Recent developments have highlighted key differences in GBP-mediated immunity between the human and mouse and how human GBPs restrict Toxoplasma remains poorly understood. In Chapter 3, I discover that macrophages induce host cell death to promote parasite clearance, and also reveal parasite restriction occurs independently of host cell death. Finally, I generated a zebrafish GBP2 knockout line by CRISPR/Cas to explore the role of GBPs in cell-intrinsic Toxoplasma control. Overall, the findings in this thesis highlight zebrafish larva as a useful model for the study of Toxoplasma infection. I use this model to reveal important roles for macrophages in Toxoplasma clearance in vivo.

Shigella flexneri is a facultative intracellular bacterium and a paradigm to address key issues in cell biology and cell-autonomous immunity. Cell-autonomous immunity is a system of host defence that senses invading pathogens and mobilises anti-pathogen mechanisms, including autophagy. Recently, it has become clear that the cytoskeleton is directly linked to cell-autonomous immunity. During my PhD, I used S. flexneri to investigate bacterial factors that mediate interactions with the cytoskeleton and cell-autonomous immunity. Bacteria have counterparts to the host cytoskeleton components actin (e.g. MreB), microtubules (e.g. FtsZ), intermediate filaments (e.g. CreS) and septins (MinCD). However, rearrangements of the bacterial cytoskeleton have never been followed in pathogenic bacteria during infection of host cells. In Chapter 1, I generate new tools to follow the Shigella MreB, FtsZ and MinC cytoskeleton during infection of host cells using fluorescence microscopy. S. flexneri can exploit the host actin cytoskeleton to form 'actin tails' for its own motility. Actin-based motility enables bacterial cell-to-cell spread and evasion of the immune system. Polar localisation of the autotransporter IcsA is required for efficient actin tail formation, yet how IcsA is targeted to the bacterial cell pole was not fully known. In Chapter 2, I use Shigella MreB-msfGFPsw to reveal that MreB targets IcsA to the bacterial cell pole to promote actin tail formation and autophagy escape. To entrap Shigella for autophagy, the host septin cytoskeleton forms cage-like structures around actin polymerising bacteria. How septins recognise bacteria is poorly understood. In Chapter 3, I report that septins sense micron-scale curvature, cardiolipin and cell growth of dividing bacterial cells to inhibit Shigella cell division via autophagy and lysosome fusion. Therefore, the host septin cytoskeleton offers great potential to boost the recognition and restriction of dividing bacterial cells. Overall, the findings in this thesis have discovered that by controlling bacterial cell polarity, morphology and division, the bacterial cytoskeleton shapes host-pathogen interactions. Moreover, they highlight that investigation of the bacterial cytoskeleton during infection can inspire the development of new therapeutic regimes for infection control.

Shigella flexneri is an invasive bacterial pathogen and inflammatory paradigm that has led to key discoveries in innate immunity. The transparent zebrafish (Danio rerio) larva is highly amenable to in vivo microscopy and has emerged as a valuable model for the study of host defence against bacterial infection. In this thesis, I use the zebrafish to explore the innate immune response to Shigella infection, and illuminate new ways to control bacterial infection in vivo. Septins are an evolutionarily conserved family of cytoskeletal proteins with diverse biological functions. Very recent work has shown a key role for components of the cytoskeleton in inflammation control, yet a role for septins was unknown. In Chapter 3, I develop a zebrafish hindbrain model of Shigella infection and reveal a new role for septins in the restriction of inflammation crucial for host defence. The global rise in antibiotic resistance amongst human pathogens has prompted an urgent search for novel antimicrobials. Bdellovibrio bacteriovorus are predatory bacteria that invade and kill Gram-negative species. Bacterial predation has been studied extensively in vitro but little is known of its efficacy in vivo. In Chapter 4, I use the Shigella-zebrafish infection model developed in Chapter 3 to show how immune cells and Bdellovibrio can work together to cure infection from multidrug resistant Shigella. Septins were discovered for their essential roles in cell division. S. flexneri is controlled by neutrophils in vivo, and emergency granulopoiesis is a haematopoietic program of neutrophil production used to counteract immune cell exhaustion during infection. In Chapter 5, I use Shigella infection of zebrafish to reveal a cell autonomous role for septins in haematopoietic stem cell-driven emergency granulopoiesis and in enhancing host immunity. Overall, the findings in this thesis have important implications for the treatment of inflammatory, infectious and haematological disease. As a result of these data, manipulation of the septin cytoskeleton represents a novel strategy by which to control inflammation. Furthermore, results obtained using Bdellovibrio are promising for the future consideration of living therapies to treat bacterial infection. Finally, the host cell division machinery can be used to modulate the haematopoietic response to infection and boost host defence. Collectively, results obtained using our Shigella-zebrafish infection model are highly translatable to higher vertebrates, including humans.

Autophagy is a highly conserved intracellular degradation process crucial for cell-autonomous immunity against bacterial infection. However, some bacteria may subvert the host cytoskeleton to escape recognition from autophagy. In the cytosol, Shigella flexneri and Listeria monocytogenes use actin-based motility to avoid cell-autonomous immunity and spread from cell-to-cell. The host cytoskeleton plays a key role in autophagy and its ability to restrict or promote bacterial replication. Septins, a cytoskeletal component that interacts with cellular membranes and actin filaments, are GTP-binding proteins that polymerize into non-polar filaments and rings. Our lab has discovered that septin cage-like structures entrap actin-polymerising Shigella targeted to autophagy. The septin cage is recognized as a mechanism of host defence mechanism, however the precise fate of septin cage entrapped Shigella remains unknown. Evidence has suggested that mitochondria provide membrane for the biogenesis of autophagosomes during starvation. The role of the mitochondria during the autophagy of Shigella is unknown. Mitochondria are highly dynamic organelles characterised by events of membrane fission and fusion. Work has previously established a role for the host cell division machinery, i.e. actin and myosin, in the regulation of mitochondrial dynamics (a process called 'mitokinesis'). Whether septins are also involved in mitochondrial dynamics has not yet been tested. For my thesis, the entrapment of Shigella by septin cages was used as a paradigm to better understand host defence against bacterial infection and to discover new septin biology. Results have shown that mitochondria mediate the assembly of septins into cage that target Shigella to degradation by autophagy. We have also discovered that septins regulate mitochondrial fission. Finally, we have revealed a new link between septins, autophagy, and host cell metabolism. Together these results suggest that a more complete understanding of septin biology during bacterial infection can enable its manipulation for therapeutic purposes.