A genome-scale census of virulence factors in the major mould pathogen of human lungs

由真菌引起的传染病是一个世界性的问题，造成的死亡人数至少与疟疾和结核病一样多。对于由孢子形成霉菌引起的感染，只有一种口服药物，氮唑。临床分离的霉菌对氮唑的耐药性正在增加，这一问题的一个可能原因是在环境中广泛使用唑类作为农业杀菌剂。因此，迫切需要开发新的抗真菌治疗方法。真菌颗粒(或孢子)持续存在于我们呼吸的空气中，能够在人类肺部存活。通常，当感染者的免疫系统没有以最佳状态运行时，就会发生这种情况。通常，它涉及真菌孢子(很像植物种子)的萌发和被称为菌丝的长真菌细胞的生长。菌丝通过分泌酶穿透和溶解肺组织。我们呼吸的空气中的主要真菌病原体称为烟曲霉，受癌症影响或需要器官移植的人特别有可能因此而受到致命感染。不幸的是，我们对烟曲霉缺乏了解，以及在与之合作时遇到的技术困难，限制了这一领域的进展。我们还不完全了解曲霉菌为什么能够在肺环境中存活。我们迫切需要关于这种病原体的更多基本信息，这样我们才能开始设计未来的治疗方法。当科学家试图弄清楚细胞过程是如何工作的时，第一个求助的往往是转录因子。转录因子同时控制许多基因的活动，因此它们在细胞过程中拥有巨大的权力。如果一个转录因子对一个特定的过程很重要，那么人们就会看看它调节了哪些基因，这将为这个过程背后的生物学提供线索。如果一个转录因子没有参与，那么它以及它调节的所有基因都可以从研究中剔除。通过询问哪些转录因子控制烟曲霉菌致病的能力，我们可以获得驱动感染的调控网络的全球视角。我们打算将这种方法与最先进的DNA测序技术相结合，以最大限度地了解烟曲霉菌的致病性，并最大限度地减少对小鼠的使用。我们搜索了烟曲霉菌基因组中的转录因子基因，并创造了401个烟曲霉突变株，每个突变株都缺少一个转录因子。为了询问哪些转录因子对哺乳动物肺部感染是重要的，我们将用一组突变基因感染小鼠，看看哪些无法在肺内存活。我们设计了一种方法，使我们能够同时测试许多变种人。这包括将所有突变体感染到一只小鼠身上，并在牺牲后，对从肺部提取的DNA进行测序，以告诉我们哪些突变体设法存活了下来。我们将用这种方法揭示所有驱动烟曲霉致病力的转录因子(P-TF)。因此，动物的数量将被尽可能少地利用。当我们确定了所有的P-TF后，我们将致力于确定由每个P-TF调控的基因。为了做到这一点，我们将使用两种方法。首先，我们将转录因子与烟曲霉菌DNA进行化学固定，以确定每个转录因子的结合部位。其次，我们将观察缺乏P-TF的突变体中的基因表达，并找出哪些基因变得不受调控。这项研究的成功将1)纠正近几十年来在确定烟曲霉致病力的分子基础方面取得的微不足道的进展2)直接引导我们进入我们需要用新药瞄准的真菌过程3)极大地减少其他科学家对烟曲霉菌进行的动物感染研究4)帮助我们优化对烟曲霉菌的高通量操作，用于未来的药物筛选。

Infectious diseases caused by fungi are a worldwide problem causing at least as many deaths as malaria and tuberculosis. For infections caused by spore-forming moulds there is only one available class of oral drug, the azoles. Azole resistance amongst clinical mould isolates is increasing and one possible cause of this problem is the widespread use of azoles in the environment as agricultural fungicides. There is therefore a desperate need to develop new antifungal treatments.Fungal particles (or spores), which are continually present in the air that we breathe, are able to survive in the human lung. Usually, this occurs when the immune system of the infected individual is not functioning optimally. Always, it involves the germination of the fungal spore (rather like a plant seed) and growth of long fungal cells called hyphae. Hyphae penetrate and dissolve lung tissues by secreting enzymes. The major fungal pathogen in the air we breathe is called Aspergillus fumigatus and people affected by cancer, or requiring organ transplants, are particularly at risk of fatal infections caused by it. Unfortunately our lack of understanding of A. fumigatus, and the technical difficulties encountered when working with it, has limited progress in this field. We do not fully understand why Aspergillus is able to survive inside the lung environment. We urgently need more basic information about this pathogen so we can begin to design future therapies. When scientists try to figure out how cellular processes work, a first port of call is often transcription factors. Transcription factors control the activity of many genes simultaneously so they command great power over cellular processes. If a transcription factor is important for a particular process, one will then look at which genes it regulates and this will give clues to the biology underlying that process. If a transcription factor is not involved then it, and all of the genes it regulates, can be eliminated from the investigation. By asking which transcription factors control the ability of A. fumigatus to cause disease, we can achieve a global view of the regulatory network which drives infection. We intend to couple this approach to a state of the art DNA sequencing technology to gain maximal insight on A. fumigatus pathogenicity, with minimal usage of mice. We have searched the A. fumigatus genome for transcription factor genes and created a collection of 401 A. fumigatus mutants, each one lacking a transcription factor. To ask which of the transcription factors is important for mammalian lung infection we will infect mice with the collection of mutants and see which are unable to survive inside the lung. We have devised a method which will allow us to test many mutants at the same time. This involves infecting all of the mutants into a single mouse, and after sacrifice, sequencing the DNA extracted from the lung to tell us which mutants have managed to survive. We will use this method to reveal all of the transcription factors driving pathogenicity (P-TFs) in A. fumigatus. Thus the lowest possible number of animals will be utilised. When we have identified all of the P-TFs we will then work to identify the genes which are regulated by each P-TF. In order to do this we will use two approaches. First we will chemically fix the transcription factor to A. fumigatus DNA to identify the binding sites of each transcription factor. Second we will look at gene expression in mutants lacking P-TFs and work out which genes become deregulated. The success of this investigation will 1) Redress the meagre progress made in recent decades in defining the molecular basis of pathogenicity of Aspergillus fumigatus 2) Lead us directly to the fungal processes we need to target with new drugs 3) Hugely reduce the number of animal infection studies performed by other scientists on A. fumigatus 4) Help us to optimise high throughput manipulation of A. fumigatus for future drug screens.