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

弓形虫是一种与疟疾寄生虫有关的病原体。它会导致脑部疾病，对免疫系统功能低下的人（包括新生儿）来说是一个严重的健康威胁。没有疫苗或药物来对抗这种疾病。弓形虫生活在细胞内一个叫做空泡的隔间里。空泡由一层膜组成，这层膜保护寄生虫免受宿主细胞防御系统的攻击。当一个生物体感染弓形虫时，身体会产生一种叫做干扰素的分子，这种分子可以刺激防御蛋白的产生。当感染产生干扰素时，称为干扰素（IFN）诱导型GTP酶的防御蛋白家族高度上调。这些IFN-诱导型GTP酶可以识别弓形虫空泡，破坏它并杀死寄生虫。这有时甚至会导致宿主细胞的自杀行为，以消除寄生虫可以生长的地方。结合起来，在整个生物体的水平上，结果是控制寄生虫和受感染的宿主存活。我们对这个系统的主要问题是：什么样的IFN-诱导型GTP酶识别液泡？它们是如何打开空泡的？到底是什么机制最终消灭了寄生虫并杀死了宿主细胞？日本的Masahiro Yamamoto小组和英国的伊娃Frickel小组将合作回答这些问题。我们将研究IFN-诱导的GTP酶如何识别膜的弓形虫空泡通过研究蛋白质直接结合脂质，形成空泡膜。我们将询问GTP酶识别哪些脂质，以及它们是否可以使膜变形。我们还将制造IFN诱导型GTP酶的敲除细胞，并研究破坏弓形虫空泡的机制是否不再起作用。如果是这样的话，那么我们将研究GTP酶的突变体，以了解它们如何打开弓形虫空泡。最后，我们将问这些GTP酶的哪些部分可以直接控制寄生虫并诱导宿主细胞死亡。为此，我们开发了一个人工智能程序，可以识别和分类我们的显微图像。接下来，我们将发现在弓形虫空泡识别和控制途径中发挥作用的新蛋白质。为此，我们将使用我们已知的具有诱饵功能的GTP酶，在发现实验中标记所有与GTP酶接近的蛋白质。这些新的蛋白质也可能靶向弓形虫空泡并打开它们或帮助诱导宿主细胞死亡。我们将使用与以前相同的方法进行研究。最后，利用所有这些信息，我们将以小鼠为模型系统，确定IFN诱导型GTP酶在真实的感染中的生理作用。我们将制造基因敲除小鼠和具有IFN诱导突变的重要突变的小鼠，并研究对弓形虫感染的免疫应答以及这些GTP酶如何影响生物体中的免疫应答。总之，我们将能够发现IFN诱导的GTP酶如何识别弓形虫空泡并对抗寄生虫的全新模式。我们还将在这条东道主防守道路上定义新的球员。只有通过相互合作，才能提供这些发现，因为两个团队都开发了互补的专业知识。我们的研究结果将有进一步的价值，以了解疾病控制的其他病原体也隐藏在液泡-这些是例如细菌，如沙门氏菌，衣原体和结核病。

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.