Defining neuron-Toxoplasma gondii interactions that mediate CNS toxoplasmosis

 描述（由申请方提供）：弓形虫对中枢神经系统的嗜性是其在发育中的胎儿、免疫功能低下者和偶尔免疫功能正常者中引起的症状性疾病的基础。作为一种细胞内寄生虫，弓形虫对宿主细胞功能的吸收能力决定了寄生虫的生存及其对宿主的影响。关于弓形虫与宿主细胞相互作用的了解主要来自于成纤维细胞和免疫细胞的体外研究。这些研究表明，弓形虫通过注射效应蛋白来控制宿主细胞的功能，这些蛋白靶向通用和宿主细胞特异性途径。虽然这些研究建立了很多关于弓形虫宿主细胞生物学的知识，但它们将错过CNS特异性的宿主细胞途径或仅在体内感染期间触发。这些限制与CNS特别相关，CNS由维持基线抗炎状态的多种相互作用的细胞类型组成。鉴于CNS-弓形虫界面对临床疾病的重要性，了解弓形虫的CNS特异性作用对于最终预防症状性神经系统疾病至关重要。为此，我们最近工程弓形虫株注射Cre重组酶到宿主细胞伴随效应蛋白。通过使用这些寄生虫感染小鼠，表达绿色荧光蛋白后，只有Cre介导的重组，我们能够永久识别和配置文件的中枢神经系统细胞注射弓形虫效应蛋白。该模型的大量初步数据强烈表明，与现有的 弓形虫在体内主要与神经元相互作用，这意味着神经元是弓形虫直接操纵的主要实质CNS细胞。本提案的目标是确定神经元-弓形虫界面如何驱动CNS感染的建立和结果。为此，我们将使用我们的新小鼠模型与两种遗传上不同的弓形虫株相结合，这两种弓形虫株导致不同的CNS结果，并且我们已经设计用于注射Cre。具体而言，使用我们开发的尖端CNS成像技术，我们将确定这些弓形虫株是否通过感染不同的CNS区域或选择不同的神经元亚型而导致不同的CNS结果（目的1）。此外，为了确定这些菌株特异性CNS效应是否继发于神经元的差异操作，我们将分离并转录这些弓形虫注射的神经元（目的2）。这项研究的成功完成将为神经元-弓形虫相互作用如何驱动CNS疾病提供第一个机制见解。最终，这些见解将为我们开发CNS特异性甚至菌株特异性疗法提供新的途径，以改善CNS疾病的结局。

 DESCRIPTION (provided by applicant): Toxoplasma gondii's tropism for the CNS underlies the symptomatic disease it causes in developing fetuses, the immunocompromised, and, occasionally, the immunocompetent. As an intracellular parasite, Toxoplasma's ability to co-opt host cell function determines both the parasite's survival and its effects on the host. What is known about the Toxoplasma-host cell interaction comes primarily from in vitro studies in fibroblasts and immune cells. These studies have revealed that Toxoplasma commandeers host cell functions in part through the injection of effector proteins and that these proteins target boh universal and host-cell-specific pathways. While these studies established much about Toxoplasma-host cell biology, they will have missed host cell pathways specific to the CNS or only triggered during in vivo infection. These limitations are particularly relevant to the CNS, which is composed of multiple interacting cells types that maintain a baseline anti-inflammatory state. Given the vital nature of the CNS- Toxoplasma interface to clinical disease, understanding the CNS-specific effects of Toxoplasma is essential to eventually preventing symptomatic neurologic disease. To this end, we recently engineered Toxoplasma strains that inject Cre recombinase into host cells concomitantly with the effector proteins. By using these parasites to infect mice that express a green fluorescent protein only after Cre-mediated recombination, we are able to permanently identify and profile CNS cells injected with Toxoplasma effector proteins. Substantial preliminary data from this model strongly suggests that, contrary to existing dogma, Toxoplasma primarily interacts with neurons in vivo, which means that neurons are the major parenchymal CNS cell directly manipulated by Toxoplasma. The goal of this proposal is to determine how the neuron-Toxoplasma interface drives the establishment and outcomes of CNS infection. To this end, we will use our new mouse model in combination with two genetically divergent strains of Toxoplasma that cause different CNS outcomes and that we have engineered to inject Cre. Specifically, using cut- ting edge CNS imaging techniques that we have developed, we will determine if these Toxoplasma strains cause distinct CNS outcomes by infecting different CNS regions or co-opting different neuron subtypes (Aim 1). Additionally, to determine if these strain-specific CNS effects are secondary to differential manipulation of neurons, we will isolate and transcriptionally profile these Toxoplasma-injected neurons (Aim 2). The successful completion of this research will provide the first mechanistic insights into how neuron-Toxoplasma interactions drive CNS disease. Ultimately, such insights will offer us new avenues for developing CNS-specific or even strain-specific therapies that improve the outcomes of CNS disease.