DEALING WITH THERAPY-RESISTANT CRYPTOCOCCOSIS BY TARGETING INTRACELLULAR PATHOGENS

隐球菌病是一种威胁生命的真菌疾病，全世界每年有60多万患者死亡。这些人中的大多数是发展中国家新诊断的艾滋病毒阳性患者，如果他们在隐球菌感染中存活下来，他们的预后相对较好。此外，在其他健康的人中，隐球菌感染的发病率在全球范围内不断上升，这似乎是由于出现了局部的、超强毒力的真菌种群。因此，更多地了解这种病原体的生物学知识，并开发出改进治疗方法是至关重要的。一个主要问题是，现有的“最佳实践”抗真菌治疗在超过10%的患者中仍然无效。其中一个原因是，在感染期间，一些隐球菌细胞‘隐藏’在患者的白细胞内。这些细胞内的真菌不会暴露在足够高水平的抗真菌药物中，因此可能会成为疾病的“蓄水池”。为了尝试和解决这个问题，我们的建议旨在确定可以用来刺激细胞内隐球菌从白细胞中排出的药物，从而使它们暴露在血液循环中致死水平的抗真菌药物中。我们的团队最近发现了这样一种排泄过程，我们称之为呕吐细胞增多症，并表明它可以通过应用不同的药物来刺激，比如抗疟疾药物氯喹。为了将这一发现应用于临床，我们需要遵循两条路线进行调查。首先，我们需要了解在受感染的白细胞内发生的分子事件，并导致真菌排出。如果我们想要设计出操纵这一过程的方法，而不会造成不必要的“附带损害”(例如，极大地削弱身体应对可能与隐球菌同时感染的其他病原体的能力)，这些信息就是必不可少的。其次，我们计划进行高通量筛选，以确定新的、更有效的刺激隐球菌排出的化合物。重要的是，我们将采取“老药新招”的方法，重点搜索已获美国食品和药物管理局批准的(&gt；1000)化合物名单。因此，这些药物符合最低安全标准，并已被用于治疗其他疾病。因此，我们确定的化合物可以比全新的分子更快、更便宜地进入临床实践。在项目结束时，我们将提供关于一种致命人类疾病的重要新信息，并揭示了一系列化合物，在不久的将来，可能会被用来帮助治疗这种疾病。

Cryptococcosis is a life-threatening fungal disease that kills more than 600 000 patients per annum worldwide. The majority of these individuals are newly diagnosed HIV-positive patients in the developing world, for whom the prognosis is relatively good, PROVIDED that they survive the cryptococcal infection. In addition, there is a globally increasing incidence of cryptococcal infection in otherwise healthy people, which appears to be due to the occurrence of localized, hypervirulent populations of the fungus. Thus understanding more about the biology of this pathogen, and developing ways to improve treatment of it, is critical.One major problem is that the 'best practice' antifungal treatment available still fails in more than 10% of patients. One reason for this is that, during an infection, some cryptococcal cells 'hide' within white blood cells of the patient. These intracellular fungi are not exposed to high enough levels of antifungal drugs and can thus act as 'reservoirs' of disease. To try and address this problem, our proposal aims to identify drugs that can be used to stimulate the expulsion of intracellular cryptococci from white blood cells, thus exposing them to lethal levels of antifungal drugs in the circulation. Our group has recently discovered just such an expulsion process, which we call vomocytosis, and shown that it can be stimulated by applying different agents, such as the anti-malarial drug chloroquine. In order to move this finding into the clinic, we need to follow two lines of investigation. Firstly, we need to understand the molecular events that happen within infected white blood cells and lead to fungal expulsion. Such information is essential if we are to devise methods of manipulating that process without causing unwanted 'collateral damage' (for instance, dramatically impairing the body's ability to tackle other pathogens that may be infecting at the same time as Cryptococcus). Secondly, we plan to undertake a high-throughput screen to identify new, more effective, compounds that stimulate cryptococcal expulsion. Importantly, we will take a 'new tricks for old drugs' approach, focusing our search on the list of (>1000) compounds that have already been approved by the Food and Drug Administration. These drugs therefore meet minimum safety standards and have already been used to treat other disease conditions. Therefore compounds that we identify can be moved into clinical practice more rapidly and more cheaply than totally new molecules.At the end of the project, we will thus have provided both important new information about a fatal human disease and revealed a set of compounds that might, in the near future, be utilized to help treat that disease.