Interferon mediated control of intracellular bacteria
Legionella pneumophila is the major causative agent of Legionnaire’s Disease, which is a vastly under diagnosed infection estimated to cause around 10% of community acquired pneumonia. Although L. pneumophila is foremost an environmental organism, the ability of the bacteria to survive and replicate in amoebae has equipped the pathogen with the capacity to survive and replicate in human alveolar macrophages. Immunocompetent hosts generally respond to L. pneumophila infection through a robust innate immune response. We and others have shown that interferon g (IFNγ) is induced early after L. pneumophila infection and is crucial for controlling bacterial load in the lung. We recently showed that IFNγ production by memory T-cells was stimulated in an antigen-independent manner and drove clearance of the bacteria from the lung by stimulating the bactericidal activity of inflammatory monocyte-derived cells (MCs). In contrast, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. The aim of this study is to investigate the molecular mechanisms by which IFNγ drives clearance of L. pneumophila from the lung. This work builds on our recent finding that monocyte derived cells are the primary responders to IFNγ production during L. pneumophila infection and that IFNγ drives a bactericidal response by upregulating the expression of guanylate binding proteins (GBP) and immunity-related GTPases (IRGs). The specific aims of this proposal are to examine the contribution of IFNγ-induced Gbp1 and Irga6 to cell autonomous defence against L. pneumophila and in vivo clearance. In this aim we will focus on the mechanisms by which Gbp1 and Irga6 which restrict L. pneumophila intracellular replication. We will express Gbp1 and Irga6 in mouse macrophages, examine their coordinated effect on bacterial survival and create genetic deletions in macrophage cell lines to determine if these cells are more susceptible to infection. In addition, we will characterise the role of other GBPs and IRGs in the restriction of L. pneumophila intracellular replication. Other GBPs and IRGs upregulated by IFNγ will be assessed for their contribution to the restriction of L. pneumophila intracellular replication following similar in vitro approaches. In addition, we will generate GBP- and IRG-deficient mice to test the relevance of our findings in vivo.
Control of innate and adaptive immunity against Legionella pneumophila by Arf-GTPase exchange factors using in vivo infection systems
Legionella pneumophila is a ubiquitous environmental bacterium, which forms a specialised phagosome, called the Legionella containing vacuole (LCV), in free-living protozoa, but Legionella can also replicate in human alveolar macrophages, dendritic cells (DC) and epithelial cells. Murine macrophages are non-permissive in principle, but may be rendered susceptible by mutations in the host genome affecting control of the LCV, and this includes genes involved in cellular metabolism and lipid kinases. The Kolanus lab has generated knock-out mouse models for all four Arf-GTPase guanine nucleotide exchange factors (GEF) of the so-called cytohesin family. Arf-GTPases are required for proper intracellular vesicle transport, cell adhesion and lipid kinase signalling. Arf1 in particular is targeted by the Legionella virulence effector, RalF, which acts as an Arf GEF and, unusually for a bacterial protein, contains a Sec7 domain. RalF functions to recruit Arf1 to the LCV. As predicted from cell biology, myeloid-specific knock-out of cytohesin- 1 or -2 yield defective cell adhesion and in vivo cell recruitment functions in macrophages, DC and in neutrophil granulocytes, respectively. However, new evidence points to an important function of these molecules in the regulation of phagocytosis and autophagy in macrophages and DC. In the course of this project, we will explore the roles of Arf GTPase regulatory pathways in both innate and adaptive immunity against Legionella infection. We will employ in vivo infection systems of knock-out mouse models with subsequent mechanistic analyses at the cell level, using dynamic subcellular imaging and biochemistry. The project will thus yield a rich educational content for aspiring students at the scientific interface of immunology, cell biology and microbiology, and will further provide highly relevant basic research for potential future public health management.