Genetic barcoding to track Toxoplasma cyst heterogeneity during brain colonization, reactivation, and drug treatment.

项目摘要 弓形虫组织包囊是人类终生慢性感染的基础。在艾滋病患者中，免疫 系统不再能控制脑囊肿复发的寄生虫，导致寄生虫失控 复制与弓形虫脑炎(TE)。由于现有药物，需要新的候选药物来治疗TE 有毒副作用，而且不能消除慢性囊肿期。目前尚不清楚目前的药物为什么会这样做 并不是消灭所有的脑囊肿。因为随着时间的推移，复发的包囊寄生虫会形成新的包囊，所以 不清楚每个囊内是否有一小部分耐药寄生虫，或者整个囊内是否有药物 抗性的或易受影响的同样，当弓形虫在免疫抑制期间重新激活并导致TE时 目前尚不清楚这是因为只有一些包囊重新激活，还是大多数包囊重新激活。因为个别的包囊 不能在池中区分，当前评估药物治疗期间或 复活依赖于以下脑囊肿总负荷的粗略测量。因此，迫切需要 开发一种方法，使人们能够跟踪大脑定植和脑囊肿的体内宿主动态 复活和药物治疗过程中的异质性。我们的初步数据显示，我们可以利用基因 条形码寄生虫跟随宿主内单个寄生虫的命运。我们的中心假设是 包囊内寄生虫的异质性决定了对治疗药物或 重新激活。在我们的第一个目标中，我们将使用表达荧光素酶的条形码寄生虫来跟踪 免疫活性小鼠的包囊形成和免疫抑制诱导的包囊再激活。这将允许 美国将确定最初接种的弓形虫疫苗的哪一部分扩散到大脑和随后的 脑囊肿随时间变化的个体水平上的动态变化。这是关键信息，将使我们能够 确定可在未来的池化屏幕中分析的功能丧失寄生虫库的复杂性 旨在识别对脑部定植、囊肿形成、囊肿持续性至关重要的寄生虫基因 以及寄生虫的复发。在这个目标中，我们还将确定哪些部分的囊泡重新激活和 传播。在我们的第二个目标中，我们将确定囊肿耐药是否是由于一小部分 单个包囊具有抗药性，或者对每个包囊内的一小部分寄生虫具有抗药性。这些结果是 预计将产生重要的积极影响，因为预计它们将允许1)更精致的 评估旨在消除慢性组织囊肿的药物的疗效；2)未来汇集的设计 功能丧失筛查以确定对脑定植、囊肿形成、囊肿形成 持久性，或寄生虫复发。

Project Summary Toxoplasma tissue cysts form the basis for lifelong chronic infections in humans. In AIDS patients, the immune system can no longer control recrudescent parasites from brain cysts leading to uncontrolled parasite replication and Toxoplasma encephalitis (TE). New drug candidates are needed to treat TE since current drugs have toxic side effects and they do not eliminate the chronic cyst stages. It is not known why current drugs do not eliminate all brain cysts. Because recrudescent parasites from cysts can form new cysts over time, it is unclear if in each cyst there is a fraction of treatment-resistant parasites or if entire cysts are either drug resistant or susceptible. Similarly, when Toxoplasma reactivates during immunosuppression and causes TE it is not known if this is due to only some cysts reactivating or if most cysts reactivate. Because individual cysts cannot be distinguished in a pool, current methods to assess cyst dynamics during drug treatment or reactivation rely on the crude measure of following total brain cyst burden. There is, therefore, a critical need to develop methodology that allows one to follow the within host-dynamics of brain colonization and brain cysts heterogeneity during reactivation and drug treatment. Our preliminary data show that we can use genetically barcoded parasites to follow the fate of individual parasites within a host. Our central hypothesis is that heterogeneity of the parasites inside cysts determines heterogeneity in resistance to therapeutics or reactivation. In our first aim, we will use barcoded parasites expressing luciferase to follow the dynamics of cyst formation in immunocompetent mice and cyst reactivation induced by immunosuppression. This will allow us to determine what fraction of the initial Toxoplasma inoculum disseminates into the brain and subsequent brain cysts dynamics, on the individual cyst level, over time. This is critical information that will allow us to determine the complexity of loss-of-function parasite libraries that can be analyzed in future pooled screens designed to identify parasite genes that are essential for brain colonization, cyst formation, cyst persistence and parasite recrudescence. In this aim we will also determine what fraction of cysts reactivates and disseminates. In our second aim, we will determine if cyst resistance to drug treatment is due to a fraction of individual cysts being resistant or to a fraction of parasites within each cyst being resistant. These results are expected to have an important positive impact because they are expected to allow 1) a more refined assessment of the efficacy of drugs designed to eliminate chronic tissue cysts; 2) the design of future pooled loss-of-function screens to identify parasite genes that are essential for brain colonization, cyst formation, cyst persistence, or parasite recrudescence.