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

人类经常接触、吞咽或吸入细菌等微生物，它们大量存在于我们周围。在绝大多数情况下，人体不会受到伤害，因为人体防御系统的特殊细胞，即巨噬细胞，会抓住入侵的细菌，吞噬并杀死它们。隔离和杀死细菌似乎是一种直接而简单的限制措施，是一个涉及许多蛋白质的高度复杂的过程，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。大多数细菌不反击，但致病菌利用被称为效应物的蛋白质来干扰抗菌机制。这场战斗的结果受到感染者健康状况的重大影响。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。我们对巨噬细胞的复杂过程以及细菌的武器的理解仍然是肤浅的；然而，需要深入的知识来设计新的治疗方法来解除细菌或增强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来研究细菌与宿主相互作用的一个新方面。嗜肺菌通常存在于环境中的淡水水体中，但如果被吸入，则在人肺巨噬细胞中存活并繁殖，导致呼吸道疾病，在老年人或免疫力低下或潜在呼吸道疾病的患者中，可发展为军团病，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（T4SS），这是一种复杂的机制，可以将数百种效应物从细菌转运到宿主细胞，在宿主细胞中，它们操纵过程以使细菌受益。为了进行这些操作，一些效应物用小的化学基团修饰宿主蛋白，即所谓的翻译后修饰（PTMs）。一些PTMs被用于人类细胞来调节修饰蛋白的活性。效应物介导的修饰可以模拟宿主细胞中通常发生的ptm，从而激活宿主蛋白的自然活性，或者是新的，抑制或重编程目标蛋白的功能。我们最近在试管中发现嗜肺乳杆菌效应物LtpM修饰了许多含有葡萄糖片段的蛋白质；然而，LtpM实际上是如何激活和附着糖的，宿主靶标的哪些残基被修饰，以及该PTM在感染过程中的作用尚不清楚。有趣的是，在哺乳动物细胞中，一个类似的，但不完全相同的一个糖片段的修饰是一个丰富的PTM，它被用来调节蛋白质的活性，以响应例如营养的可用性。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，我们的目标是通过确定其三维结构和鉴定其功能所必需的残基来揭示LtpM的作用模式。我们将分析被LtpM和/或管家机制修饰的蛋白质，并分析这两种PTMs是共存还是竞争。然后，我们将确定ptm对修饰蛋白活性的影响及其在嗜肺乳杆菌感染中的作用，有望揭示新的细菌战策略和关于人类宿主反应的基本新知识，为了解感染易感性和设计新的抗菌疗法提供完整的信息。

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.