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

项目摘要 弓形虫组织囊肿构成了人类终身慢性感染的基础。在艾滋病患者中， 系统无法再控制从脑囊肿复发的寄生虫，导致寄生虫失控 复制和弓形虫脑炎（TE）。需要新的候选药物来治疗TE，因为目前的药物 具有毒副作用，并且它们不能消除慢性囊肿阶段。目前尚不清楚为什么目前的药物 不能消除所有的脑囊肿因为从囊肿复发的寄生虫可以随着时间的推移形成新的囊肿， 不清楚每个囊肿中是否有一小部分耐药寄生虫，或者整个囊肿是否是药物 抵抗或易感。同样，当弓形虫在免疫抑制期间重新激活并引起TE时， 目前尚不清楚这是由于只有一些囊肿重新激活或如果大多数囊肿重新激活。因为单个囊肿 不能在池中区分，目前评估药物治疗期间囊肿动力学的方法，或 再激活依赖于以下总脑囊肿负荷的粗略测量。因此，迫切需要 发展一种方法，使人们能够遵循脑定植和脑囊肿的宿主内动力学 在再激活和药物治疗期间的异质性。我们的初步数据显示，我们可以利用基因 条形码寄生虫，以跟踪个体寄生虫在宿主内的命运。我们的核心假设是， 包囊内寄生虫的异质性决定了对治疗药物的抗性的异质性， 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 免疫活性小鼠中的囊肿形成和免疫抑制诱导的囊肿再激活。这将允许 我们来确定最初弓形虫接种物的哪一部分传播到大脑中， 脑囊肿动态，在单个囊肿水平上，随着时间的推移。这是一个重要的信息，可以让我们 确定功能丧失型寄生虫文库的复杂性，以便在未来的合并筛选中进行分析 旨在确定寄生虫基因是必不可少的大脑殖民，囊肿形成，囊肿持久性 和寄生虫复发在这个目标中，我们还将确定什么样的囊肿重新激活， 传播。在我们的第二个目标中，我们将确定囊肿对药物治疗的抵抗是否是由于一小部分的 单个孢囊是抗性的，或者每个孢囊内的一部分寄生虫是抗性的。这些结果 预计将产生重要的积极影响，因为他们预计将允许1）更完善的 评估旨在消除慢性组织囊肿的药物的疗效; 2）设计未来的合并 功能丧失筛选，以确定对脑定植、囊肿形成、囊肿形成和囊肿形成至关重要的寄生虫基因。 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.