Control of genomic integrity and virulence of Aspergillus fumigatus by ADP-ribosylation.

尽管真菌能够引起严重的危及生命的疾病，但它们对世界范围内人类健康的重大影响往往被忽视。然而，真菌感染导致的死亡人数超过疟疾或结核病。烟曲霉是我们每天吸入的一种常见霉菌，是人类疾病的主要来源。随着脆弱患者人数的增加和耐药烟曲霉变种的出现，危及生命的真菌感染的威胁正在上升。为了解决这个问题并开发新的治疗策略，我们必须了解曲霉菌如何在患者体内生存。人类免疫系统通过破坏入侵病原体的DNA来对抗真菌感染，从而严重限制微生物在体内的生存能力。因此，致病微生物已经开发出一种复杂的DNA修复机制，以在宿主体内生存。然而，在真菌病原体中，识别受损DNA的机制、招募的修复因子以及避免基因组灾难的机制还不是很清楚。我的工作重点是一种名为ADP-核糖化的细胞信号，它标记DNA损伤的位置并吸引修复机制。通过研究这个信号，我的工作将为曲霉如何防御宿主提供宝贵的见解，可能使我们能够设计新的疗法来治疗耐药真菌。基于强大的数据，我的假设是，ADP-核糖化是协调对DNA损伤的反应和修复的关键信号。然而，我们目前还不清楚1)信号是如何响应DNA损伤产生的，2)它是如何协调修复机械组装的，或者3)信号是否需要在宿主中生存。为了解决这个问题，我将回答以下问题：1)相关的信号蛋白是什么样子的？观察这些信号蛋白的3D结构将有助于我们了解它们是如何工作的。这很有挑战性，因为这些蛋白质大约比一粒大米小一百万倍。我将通过使用一种名为X射线结晶学的成熟技术来克服这一挑战。这使我们能够在复杂的计算机软件的帮助下，根据晶体样品发出的光的图案来确定目标蛋白质的3D结构。了解信号蛋白的工作原理将有助于我们解决它们在真菌细胞中如何发挥作用的问题，并在未来使我们能够设计治疗感染的新药。2)ADP-核糖化如何调节DNA修复？由于DNA完整性和ADP核糖化信号对真菌的存活都是必不可少的，我们必须能够在细胞内的特定时间控制这两种信号。为了实现这一点，我将使用最先进的技术，利用光来诱导DNA损伤或去除蛋白质。然后，我会比较有和没有信号能力的真菌，提出这样的问题：没有信号，修复可以发生吗？在修复后，由于无法移除信号，真菌对持续的紧急状态有何反应？ADP-核糖化信号对于不同类型的DNA损伤是否不同？3)ADP-核糖化如何支持真菌在宿主中的存活？微生物与宿主的相互作用极其复杂，因此，从宿主外研究真菌获得的见解并不总是解释感染期间发生的事情。因此，我将使用可培养的免疫细胞和动物模型，它们具有与人类相似的感染反应，以了解在感染的哪个阶段ADP-核糖化信号是活跃的，以及削弱它是否对宿主有利。总而言之，这项研究将为真菌DNA修复带来令人兴奋的新见解，从而帮助我们了解这些危险的病原体如何克服宿主防御的一个关键方面。这可能导致抗击危及生命的真菌感染的新治疗策略。

Despite their ability to cause serious life-threatening illnesses, the major impact of fungi upon human health world-wide is often overlooked. However, fungal infections kill more people than malaria or tuberculosis. Aspergillus fumigatus, a common mould that we inhale every day, is a primary source of human disease. The threat of life-threatening fungal infections is rising with the increase in the vulnerable patient population and the appearance of drug resistant Aspergillus fumigatus variants. To overcome this problem and develop new treatment strategies, we must understand how Aspergillus can survive in a patient's body.The human immune system combats fungal infections partly by damaging the DNA of the invasive pathogen, thus severely restricting the microbe's ability to survive within the body. Therefore, disease-causing microbes have developed a sophisticated DNA repair machinery to survive in their hosts. However, in fungal pathogens, the mechanisms by which damaged DNA is recognised, the repair factors recruited, and a genomic catastrophe avoided are not well understood. My work focuses on a cellular signal termed ADP-ribosylation, which marks locations of DNA damage and attracts the repair machinery. By studying this signal, my work will provide invaluable insights into how Aspergillus defends itself against the host, potentially enabling us to design new therapies to treat drug-resistant fungi.Based on strong data, my hypothesis is that ADP-ribosylation is a crucial signal that coordinates the response to, and repair of, DNA damage. However, we do not currently understand 1) how the signal is generated in response to the DNA damage, 2) how it coordinates the repair machinery assembly, or 3) whether the signal is required to survive in the host. To address this, I will answer the following questions:1) What do the relevant signalling proteins look like? Seeing the 3D structure of these signalling proteins will help us understand how they work. This is challenging, because these proteins are about one million times smaller than a grain of rice. I will overcome this challenge by using a well-established technique called X-ray crystallography. This allows us - with the help of sophisticated computer software - to determine the 3D structure of target proteins from the patterns of light veering off a crystalline sample. Understanding the workings of the signalling proteins will help us address questions of how they function within fungal cells and in the future enable us to design novel drugs to treat infections.2) How does ADP-ribosylation regulate DNA repair? Since both DNA integrity and ADP-ribosylation signalling are essential for fungal survival, we must be able to control both at specific times within cells. To achieve this, I will use state-of-the-art techniques utilising light to induce DNA damage or remove proteins. I will then compare fungi with and without the ability to signal, asking questions like: Can repair happen without the signal? How does the fungus react to a constant state of emergency due to an inability to remove the signal after the repair? Does ADP-ribosylation signalling differ for different types of DNA damage?3) How does ADP-ribosylation support fungal survival in the host? The interplay of a microbe with its host is extremely complex, and thus insights gained from studying fungi outside the host cannot always explain what occurs during an infection. Therefore, I will use culturable immune cells and animal models, which have an infection response comparable to humans, to understand at which stages of infection ADP-ribosylation signalling is active and whether impairing it has beneficial outcomes for the host.Overall, this study will bring exciting new insights into fungal DNA repair, thus helping us to understand how these dangerous pathogens overcome a crucial aspect of host defence. This is likely to lead to new therapeutic strategies to combat life-threatening fungal infections.