Dissecting the role of infection-driven protein mono-glycosylation in Legionella-host interaction

人类不断地接触、吞咽或吸入细菌等微生物，这些微生物在我们周围的任何地方都大量存在。在绝大多数这些遭遇中，身体仍然没有受到伤害，因为身体防御的专门细胞，所谓的巨噬细胞，捕捉细菌入侵者，摄取并杀死它们。表面上看起来是一种直接和简单的限制性措施，隔离和杀死细菌是一个高度复杂的过程，涉及许多蛋白质，这些蛋白质重塑巨噬细胞以吞噬细菌并触发细胞内机制来拆除它们。大多数细菌不会反击，但病原菌利用蛋白质，称为效应子，干扰抗菌机制。这场战斗的结果在很大程度上受感染者的健康状况影响。随着年龄的增长或潜在的健康状况以及由于不良的生活方式选择，如吸烟，巨噬细胞执行这种基本保护功能的能力减弱，使病原体更容易压倒它们并引起疾病。然而，需要深入的知识来设计新的治疗方法，以解除细菌的武装或提高巨噬细胞的抗菌能力。在本项目中，我们将以嗜肺军团菌为模型，研究细菌与宿主相互作用的新方面。嗜肺军团菌通常存在于环境中的淡水水体中，但如果吸入，则会在人体肺部巨噬细胞中存活并繁殖，导致呼吸系统疾病，而老年人或免疫力受损或患有潜在呼吸系统疾病的患者可能发展为退伍军人症，这是一种可能致命的肺炎。巨噬细胞的利用需要Dot/Icm IV型分泌系统（T4 SS），这是一个复杂的机制，它将数百种效应物从细菌转运到宿主细胞中，在其中它们操纵过程以使细菌受益。为了进行这些操纵，一些效应物用小的化学基团修饰宿主蛋白，所谓的翻译后修饰（PTM）。一些PTM用于人细胞中以调节修饰蛋白的活性。效应子介导的修饰可以模拟通常发生在宿主细胞中的PTM，从而激活宿主蛋白的天然活性，或者是新的，抑制或重编程靶蛋白的功能。嗜肺菌效应子LtpM用葡萄糖部分修饰许多蛋白质;然而，LtpM实际上如何激活和附着糖，宿主靶标的哪些残基被修饰以及这种PTM在感染期间的作用仍然未知。有趣的是，在哺乳动物细胞中，具有一个糖部分的类似但不相同的修饰是丰富的PTM，并且被管家蛋白用于响应于例如营养物质的可用性来调节蛋白质活性。它与糖尿病等疾病有关;然而，它在人体细胞对细菌感染的反应中的作用尚未得到很好的表征。在这个项目中，我们的目标是揭示LtpM的作用方式，通过确定其三维结构和识别残基，这是必不可少的功能。我们将分析由LtpM和/或管家机制修饰的蛋白质，并分析这两种PTM是否共存或竞争。然后我们将确定PTM对修饰蛋白活性的影响及其在L. pneumophila感染，有望揭示一种新的细菌战策略和关于人类宿主反应的基本新知识，了解感染易感性和设计新的抗微生物疗法的完整信息。

Humans constantly touch, swallow or inhale microorganisms such as bacteria, which exist in large numbers everywhere around us. In the vast majority of these encounters the body remains unharmed, because specialised cells of the body's defences, so called macrophages, catch the bacterial intruders, ingest and kill them. Seemingly a straight forward and simple restrictive measure, sequestration and killing of the bacteria is a highly complex process involving many proteins, which reshape the macrophage to engulf the bacteria and trigger intracellular mechanisms to dismantle them. Most bacteria do not fight back, but pathogenic bacteria employ proteins, termed effectors, to interfere with antimicrobial mechanisms. The outcome of this battle is significantly influenced by the health status of the infected person. With increasing age or underlying health conditions as well as due to poor life style choices such as smoking the capacity of macrophages to carry out this essential protective function diminishes, making it easier for pathogens to overwhelm them and cause disease.Our understanding of the complex processes in the macrophage as well as of the weaponry of the bacteria is still superficial; however, in depth knowledge will be required to design new treatments to disarm the bacteria or boost the antimicrobial power of macrophages. Here, in this project we will use the bacterium Legionella pneumophila as model to investigate a new aspect of the interaction of bacteria and host.L. pneumophila is usually found in fresh water bodies in the environment, but if inhaled survives and multiplies in human lung macrophages causing respiratory disease, which in the elderly or patients with compromised immunity or underlying respiratory disease can develop into Legionnaires' disease, a potentially fatal pneumonia. The exploitation of macrophages requires the Dot/Icm type IV secretion system (T4SS), a complex machinery that transports hundreds of effectors from the bacteria into the host cell, in which they manipulate processes to the benefit of the bacteria.To carry out these manipulations some effectors modify host proteins with small chemical groups, so called post-translational modifications (PTMs). Some PTMs are used in human cells to modulate the activity of the modified protein. The effector-mediated modifications can mimic PTMs usually occurring in host cells, thus activating a natural activity of the host protein, or be new, inhibiting or reprogramming the function of the target protein.We recently discovered that in the test tube the L. pneumophila effector LtpM modifies numerous proteins with a Glucose sugar moiety; however, how LtpM actually activates and attaches the sugar, which residues of the host targets are modified and what the role of this PTM during infection is remains unknown. Interestingly, in mammalian cells a similar, but not identical modification with one sugar moiety is an abundant PTM and used by house-keeping proteins to modulate protein activity in response to for example nutrient availability. It has been implicated in diseases such as diabetes; however, its role in the response of human cells to bacterial infection is not well characterised yet. In this project we aim to reveal the mode of action of LtpM by determining its three dimensional structure and identifying residues, which are essential for its function. We will profile the proteins that are modified by LtpM and/or the house-keeping machinery and analyse if these two PTMs co-exist or compete. We will then determine the effect of the PTMs on the activities of the modified proteins and their role in L. pneumophila infection, promising to reveal a new bacterial warfare strategy and fundamental new knowledge about the human host response, integral information for understanding susceptibility to infection and designing new antimicrobial therapies.