Epigenetics of stress-induced genome instability in the human fungal pathogen Candida albicans

白色念珠菌是一种机会真菌(酵母的一种形式)，通常生活在人体内，不会造成任何伤害。然而，白色念珠菌可以导致毁灭性的疾病，特别是在接受器官移植、化疗或艾滋病毒治疗的免疫功能低下的患者中。在感染期间，白色念珠菌在我们身体的几个部位定居，因此需要在身体内的许多不同环境中茁壮成长。例如，致病的白色念珠菌在高温(即发烧)中存活，耐药的白色念珠菌菌株在抗真菌药物的存在下茁壮成长。这项提议的主要目标是回答一个关键问题：白色念珠菌如何适应如此不同的环境？在所有活着的有机体中，基因组包含构建有机体并使其生长发育所需的信息(基因)。在大多数生物中，必须维持基因组组织，以确保基因的正确平衡(从而确保使我们的身体正常运作的指令)。这在白色念珠菌中是不同的：这种真菌，像许多其他人类真菌病原体一样，具有灵活的基因组。这意味着它的基因组结构发生了变化，白念珠菌可以在没有合适比例的基因的情况下生存，甚至失去一部分染色体。重要的是，当白色念珠菌遇到恶劣环境(例如药物治疗)时，白色念珠菌的基因组灵活性会增强，这使得可以选择具有允许在这些恶劣环境中生存的基因组合的新基因组组织。我的实验室发现，基因组的灵活性源于特定DNA位点的DNA断裂：tre热点。在白色念珠菌的每个染色体上都发现了相同的tre位点，因此，一旦断裂，tre位点就可以相互作用和融合。因此，白念珠菌基因组可以自我重排，导致一些序列丢失和其他序列重复。它还可以将基因组的两个独立部分结合在一起。我实验室的工作已经证明，tre位点的DNA包装(染色质)可以引导或阻止tre热点的断裂。目前尚不清楚是什么使tre位点特别。这是不可能预测的，因为在其他生物体中没有发现Tre热点，因此不可能猜测它们的功能。在这项提案中，我们将利用我们在染色质和白色念珠菌生物学方面的专业知识来回答三个关键问题：1.为什么DNA断裂是tre位点？我们将使用遗传学和基因组学来确定为什么tre热点是DNA断裂位点。2.DNA包装是如何控制Tre断裂的？我们假设Tre热点的DNA包装在不同的环境中会发生变化。这使得Tre热点暴露在恶劣的环境中，并在不受压力的牢房中受到保护。3.tre突变的后果是什么？我们将使用CRISPR/Cas9基因组编辑来生成一个携带所有可能的tre基因组改组组合的白色念珠菌菌株文库。我们将测试这些基因组组织中哪些有利于白念珠菌的毒力(即它导致感染的效率)。了解tre热点是如何工作的，将有助于制定阻止基因组灵活性、阻止感染和耐药性的策略。重要的是，在人类中没有发现tre DNA序列，因此阻止这些位置的DNA改组不会对我们的身体产生任何有害影响。

Candida albicans is an opportunistic fungus (a form of yeast) that normally lives on the human body without causing any harm. However, C. albicans can cause devastating diseases especially in immunocompromised patients who have undergone organ transplants, chemotherapy, or HIV treatment. During infections, C. albicans colonises several parts of our body and therefore needs to thrive in the many different environments found in the body. For example, pathogenic C. albicans survives high temperatures (i.e. fever) and drug resistant C. albicans strains thrive in the presence of anti-fungal drugs. The main goal of this proposal is to answer the key question: How can C. albicans adapt to such different environments?In all living organisms, the genome contains the information (genes) needed to build the organism and allow it to grow and develop. In most organisms, genome organisation must be maintained to ensure the right balance of genes (and therefore of the instructions for making our body function correctly).This is different in C. albicans: this fungus, like many other human fungal pathogens, has a flexible genome. This means that its genome structure changes and that C.albicans can live without the right proportion of its genes or even lose a part of a chromosome. Importantly, C. albicans' genome flexibility is enhanced when C. albicans encounters hostile environments (such as for example drug treatments) this allows selection of new genome organisations with a combination of genes that allow survival in these hostile environments. My laboratory has discovered that genome flexibility originates by breaking the DNA at specific DNA sites: the TRE hotspots. Identical TRE sites are found at each of C. albicans chromosomes and therefore, once broken, TRE sites can interact and fuse with each other. As a result, the C. albicans genome can rearrange itself, causing loss of some sequences and duplication of others. It can also bring two separate sections of the genome together. Work from my laboratory has demonstrated that the DNA packaging (chromatin) of TRE sites can direct or stop breaks at TRE hotspots.It is unknown what makes TRE sites special. This is impossible to predict because TRE hotspots are not found in other organisms and therefore it is not possible to guess their function. In this proposal, we will use our expertise in chromatin and C. albicans biology to answer 3 key questions:1. Why are TRE sites of DNA breaks?We will use genetics and genomics to determine why TRE hotspots are sites of DNA breakage. 2. How does the DNA packaging control TRE breaks?We hypothesise that the DNA packaging of TRE hotspots changes in different environments. This allows TRE hotspots to be exposed in hostile environments and to be protected in unstressed cells. 3. What are the consequences of TRE breaks?We will use CRISPR/Cas9 genome editing to generate a library of C. albicans strains carrying all the possible combination of TRE genomic shuffling. We will test which of these genomic organisation are beneficial for C. albicans virulence (ie how efficiently it causes infection).Understanding how TRE hotspots work will help the development of strategies to block genome flexibility stopping infections and drug resistance. Importantly, TRE DNA sequences are not found in humans and therefore blocking DNA shuffling at these sites will not have any deleterious effect in our body.