Biophysical and physiological characterisation of potassium channels from pathogenic fungi

在免疫系统受损的人中，机会性真菌病原体是威胁生命的感染的主要原因。在过去二十年中，面临严重真菌感染风险的患者人口增加，包括艾滋病毒/艾滋病患者、接受血液和骨髓移植、大手术或接受化疗的患者，导致侵袭性感染的频率相应增加。由于真菌和它们的哺乳动物宿主之间的生物学相似性，有针对性的抗真菌治疗往往是复杂的。此外，真菌感染可能对治疗顽固不化，对氟康唑等传统干预措施的耐药性是一个日益严重的问题。因此，确定新的抗真菌靶标的必要性是至关重要的。本研究项目的目的是表征真菌离子通道蛋白及其在真菌细胞中的作用。离子通道在细胞膜上起到了调节作用，允许钠和钾等离子进出细胞，它们的功能对于维持几乎所有生命形式的细胞活动都是必不可少的。离子通道可以简单地根据它们的结构和它们通过的离子来定义。例如，钾离子通道只允许钾进入或流出细胞，并由围绕着一个“空洞”或“离子通道”的多个蛋白质组装而成。这条通路的开启和关闭受到多种刺激的严格调控。因此，了解这些离子通道蛋白如何打开和关闭以及如何刺激或抑制钾离子通量的机制是操纵真菌细胞功能的基础。利用几种人类致病真菌(白色念珠菌、烟曲霉和新生隐球菌)，这些真菌代表了免疫抑制人群中致命感染的主要来源，我已经确定了一些特定的钾通道(称为TOK1)。这些TOK1钾通道通过管理钾离子在细胞内和细胞外的运动，潜在地控制真菌细胞的生长和功能。更重要的是，如果离子通道保持开放，离子流动不受控制，可能会导致过量的钾流失和真菌细胞死亡。TOK1通道只在真菌中发现，而在人类、动物或植物中不存在类似的蛋白质。由于现有治疗方法的局限性之一是它们不区分人类和真菌细胞，这些TOK1通道的独特真菌性质使它们成为未来抗真菌治疗的理想靶点。这项研究的目的是了解这些通道如何打开和关闭的机制，并开始解决这些通道在真菌细胞中所起的作用。预计这项研究的数据将使未来能够设计出专门针对通过TOK1通道诱导不受调节的钾流动的化合物，从而提供一种独特的战略来对抗和减少危险的真菌感染的流行。

Opportunistic fungal pathogens are a major cause of life-threatening infections in individuals with a compromised immune system. An increase in the patient population at risk from the development of serious fungal infections, including HIV/AIDS patients, those undergoing blood and marrow transplant, major surgery or receiving chemotherapy has led to an associated rise in the frequency of invasive infections over the past two decades. Targeted anti-fungal therapies are often complicated by biological similarities between fungi and their mammalian hosts. Furthermore, fungal infections can be recalcitrant to therapy and resistance to traditional interventions such as fluconazole is a growing problem. Hence the need to identify novel anti-fungal targets is paramount.The purpose of this research project is to characterise a fungal ion channel protein and its role in the fungal cell. Ion channels act as regulated "holes" in the cell membrane allowing ions such as sodium and potassium to pass in and out of the cell and their function is essential for maintaining the activity of cells in almost all forms of life. Ion channels can be defined simply on the basis of their structure and which ions they pass. Potassium ion channels for example allow only the movement of potassium into or out of the cell and are formed through the assembly of multiple proteins surrounding a "hole" or "ion pathway". The opening and closing of this pathway is tightly regulated by a variety of stimuli. Thus understanding the mechanisms of how these ion channel proteins open and close and how we could stimulate or inhibit potassium ion flux is fundamental to manipulating fungal cell function. Using several species of human pathogenic fungi (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans) which represent the primary sources of fatal infections in the immunosuppressed population, I have identified a number of specific potassium channels (called TOK1). These TOK1 potassium channels potentially control the growth and function of fungal cells by managing the movement of potassium ions in and out of the cell. More importantly, if the ion channel pathway was held open and ion flow unregulated it could lead to excessive potassium loss and fungal cell death. TOK1 channels are found only in fungi and no similar protein exists in humans, animals or plants. As one of the limitations of existing therapies is that they do not distinguish between human and fungal cells, the unique fungal nature of these TOK1 channels makes them ideal targets for future anti-fungal therapies. The aim of this study is to understand the mechanism of how these channels open and close and begin to address the role these channels play in fungal cells. It is anticipated that data from this study will allow the future design of compounds specially targeted to induce unregulated potassium flow through TOK1 channels providing a unique strategy to combat and reduce the prevalence of dangerous fungal infections.