Structure-function analysis of a pH-responsive molecular switch required for fungal pathogenicity

大多数真菌都非常有用，例如，抗生素、面包、葡萄酒和啤酒的生产都涉及真菌。然而，就像细菌和病毒一样，真菌也会导致危及生命的疾病。与细菌和病毒不同，真菌细胞与人类细胞有许多相似之处。因此，很难找到既能杀死真菌又不会对人类病人造成伤害的药物。真菌引起的传染病是一个世界性的问题，最近的估计表明，真菌感染造成的死亡人数至少与疟疾和结核病一样多。目前治疗真菌感染的药物只有三类，而且它们都有局限性。该研究项目将对一组真菌蛋白进行表征，这些蛋白质已被证明是哺乳动物宿主生存所必需的，目的是寻找抑制它们的药物。我们将重点关注一类特别适合于药物发现的信号蛋白，即G蛋白偶联受体（gpcr）。真菌细胞使用gpcr样蛋白收集周围环境的信息，因此很可能已经存在抑制这些蛋白的化学物质，但在传统的药物筛选方法中被忽视了。目前获得许可的药物中大约有一半是针对gpcr的。gpcr样蛋白位于细胞表面，具有由7个跨膜片段组成的特征结构。gpcr是一种感觉分子，它检测细胞外的信号，并通过细胞内信号分子传递这些信息，从而引发细胞反应。gpcr作为药物靶点的成功是由于它们位于细胞表面，蛋白质中存在可被药物阻断的重要口袋或折叠，以及多种技术和工具的可用性来监测gpcr介导的信号传导。大多数真菌使用一种类似gpcr的蛋白质（称为PalH或Rim21）来感知周围环境的pH值。在致病性真菌中，PalH或与其通信的转录因子（PacC）的缺失会阻止真菌在哺乳动物宿主中的存活。最近，在人类的主要霉菌病原体烟曲霉中，我们发现pH信号的丧失影响了与真菌感染相关的多个过程，包括细胞壁生物合成和肺内膜的渗透。此外，我们发现pH信号缺陷的突变体极易被棘白菌素抗真菌药物杀死，而这些药物通常无法杀死曲霉。当我们在哺乳动物感染模型中测试这一观察结果时，我们发现在棘白菌素治疗的小鼠中，pH非信号突变体被清除得更有效。因此，单独或与现有抗真菌药物联合，抑制真菌pH信号可能是一种有价值的治疗选择。在这项研究中，我们将发现PalH传感器如何通过系统地突变传感器来检测细胞外pH值。阻止pH信号传导的突变将告诉我们蛋白质的哪一部分需要信号传导。此外，我们将尝试在实验室中制造足够的PalH蛋白，以形成晶体并分析蛋白质结构。在实践中，膜蛋白结构的分辨率一直非常具有挑战性，但最近的方法进步提高了成功率，并且获得蛋白质结构的3D模型将为蛋白质和可能的抑制剂的计算分析提供许多可能性。最后，根据结构和突变分析，我们将开发使用荧光蛋白的检测方法，这将有助于高通量筛选PalH抑制剂。这项研究产生的工具和见解将为抗真菌药物的发现开辟新的途径。在真菌基因组中，有数百种gpcr样蛋白被编码，但迄今为止被表征的数量不到1%。

Most fungi are extremely useful, for example, the production of antibiotics, bread, wine, and beer all involve fungi. However, like bacteria and viruses, fungi can cause life-threatening diseases. Unlike bacteria and viruses, fungal cells share many similarities with those of humans. For this reason it has been very difficult to identify drugs which will kill fungi, without causing harm to the human patient. Infectious diseases caused by fungi are a worldwide problem and recent estimates suggest that fungal infections cause at least as many deaths as malaria and tuberculosis. There are currently only three classes of drugs with which to treat fungal infections, and there are limitations associated with all of them. This research project will characterize a group of fungal proteins, already proven to be required for survival in the mammalian host, with a view to finding drugs which inhibit them. We will focus upon a specific class of signaling protein which is outstandingly amenable to drug discovery, known as G protein coupled receptors (GPCRs). Fungal cells use GPCR-like proteins to gather information about their surroundings, it is therefore likely that chemistries to inhibit these proteins already exist but have been overlooked in conventional drug screening approaches.Roughly half of currently licensed drugs target GPCRs. GPCR-like proteins are located at the surface of the cell, and have a characteristic structure composed of seven membrane-spanning segments. GPCRs are sensory molecules which detect signals outside of the cell, and convey this information via intracellular signaling molecules, to elicit a cellular response. The success of GPCRs as drug targets is due to their location at the surface of the cell, the existence of important pockets or folds in the proteins which can be blocked by drugs, and the availability of multiple technologies and tools to monitor GPCR-mediated signaling.Most fungi use a GPCR-like protein (called PalH or Rim21) to sense the pH of their surroundings. In pathogenic fungi the loss of PalH, or the transcription factor (PacC) it communicates with, prevents fungal survival in the mammalian host. Recently, in the major mould pathogen of humans, Aspergillus fumigatus, we found that loss of pH signalling impacts multiple processes associated with fungal infection including cell wall biosynthesis and penetration of the lung lining. Additionally we found that mutants defective in pH signaling became highly susceptible to killing by echinocandin antifungal drugs, which are usually unable to kill Aspergillus species. When we tested this observation in a mammalian infection model, we found that pH non-signaling mutants were cleared much more efficiently in echinocandin-treated mice. Thus alone, and in combination with existing antifungal drugs, inhibition of fungal pH signaling could be a valuable treatment option. During this research we will find out how PalH sensors detect extracellular pH by systematically mutating the sensor. Mutations which prevent pH signaling will inform us upon which segments of the protein are required for signaling. Additionally we will try to make enough of the PalH protein in the laboratory to allow formation of crystals and analysis of the protein structure. In practice the resolution of membrane protein structure has been very challenging but recent methodological advances have improved success rates and access to a 3D model of protein structure will unlock many possibilities for computational analysis of the protein and possible inhibitors. Finally, informed by the structural and mutational analyses, we will develop assays using fluorescent proteins, which will be useful for high throughput screening of PalH inhibitors. The tools and insight generated by this research will open new avenues for antifungal drug discovery. In fungal genomes many hundreds of GPCR-like proteins are encoded but to date the number characterized is less than 1 per cent.