Elucidation of cell-autonomous immunity and host defense against vacuolar pathogens by interferon-inducible GTPases

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Toxoplasma gondii is a pathogen related to the malaria parasite. It can cause brain disease and is a serious health threat to individuals with a poorly functioning immune system, including newborns. No vaccines or drugs to fight the disease are available. Toxoplasma lives inside cells in a compartment called a vacuole. The vacuole is made up of a membrane and this shields the parasite from the defence system of the host cell. When an organism is infected with Toxoplasma, the body generates molecules called interferons, which can stimulate the production of defence proteins. A family of defence proteins called the interferon (IFN)-inducible GTPases are highly upregulated when infections produce interferons. These IFN-inducible GTPases can recognise the Toxoplasma vacuole, destroy it and like this kill the parasite. This can also sometimes even lead to the death of the host cell in a suicide act to eliminate the place the parasite can grow in. In combination, on the level of the whole organism, the result is the the control of the parasite and the infected host surviving. Major questions we have about this system are: What do IFN-inducible GTPases recognise on the vacuole? How do they open the vacuoles? Exactly what mechanisms eventually eliminates the parasite and kills the host cell? The Japanese group of Masahiro Yamamoto and the UK group of Eva Frickel will team up to answer these questions. We will study how IFN-inducible GTPases recognise the membrane of the Toxoplasma vacuole by studying the proteins directly in combination with the lipids that form the vacuole membrane. We will ask what lipids are recognised by the GTPases and if they can deform the membrane. We will also make knockout cells of IFN-inducible GTPases and investigate if the mechanism of breaking the Toxoplasma vacuole does not function any more. If this is the case, then we will study mutants of the GTPases to understand how they open the Toxoplasma vacuoles. Finally, we will ask which parts of these GTPases can directly control the parasite and induce host cell death. For this, we have developed an artificial intelligence program that can recognise and classify our microscopy images. Next, we will discover new proteins that play a role in the Toxoplasma vacuole recognition and control pathway. For this we will use the GTPases we know to have a function as bait in a discovery experiment that marks all proteins close to the GTPases. These new proteins can potentially also target the Toxoplasma vacuoles and open them or help induce the host cell death. We will study this using the same methods as before. Finally, with all this information, we will determine the physiological role of IFN-inducible GTPases in a real infection using the mouse as a model system. We will make knockout mice and mice with important mutations in IFN-inducible mutations and study the immune response to a Toxoplasma infection and how these GTPases influence this in an organism.In summary, we will be able to find totally novel patterns of how IFN-inducible GTPases recognise Toxoplasma vacuoles and fight the parasite. We will also define new players in this host defence pathway. Only by teaming up with each other, will be able to deliver these findings as both teams have developed complementary expertise. Our findings will have further value to understand disease control of other pathogens that also hide in vacuoles - these are for example bacteria such as Salmonella, Chlamydia and Tuberculosis.