Defining neuron-Toxoplasma gondii interactions that mediate CNS toxoplasmosis

 描述(由申请人提供)：弓形虫对中枢神经系统的嗜性是它在发育中的胎儿中引起的症状性疾病的基础，免疫功能受损，偶尔也会免疫活性。作为一种细胞内寄生虫，弓形虫利用宿主细胞功能的能力决定了寄生虫的生存及其对宿主的影响。关于弓形虫-宿主细胞相互作用的已知主要来自于对成纤维细胞和免疫细胞的体外研究。这些研究表明，弓形虫侵占宿主细胞功能的一部分是通过注射效应蛋白，这些蛋白针对BoH通用和宿主细胞特有的途径。虽然这些研究建立了许多关于弓形虫宿主细胞生物学的基础，但它们可能错过了中枢神经系统特有的宿主细胞途径，或者只在体内感染时触发。这些限制与中枢神经系统尤其相关，中枢神经系统由多种相互作用的细胞类型组成，维持基线抗炎状态。鉴于中枢神经系统-弓形虫与临床疾病接口的重要性质，了解弓形虫对中枢神经系统的特定影响对于最终预防症状性神经疾病至关重要。为此，我们最近改造了弓形虫菌株，将Cre重组酶与效应蛋白一起注射到宿主细胞中。通过使用这些寄生虫感染只有在Cre介导的重组后才表达绿色荧光蛋白的小鼠，我们能够永久地鉴定和分析注射了弓形虫效应蛋白的CNS细胞。来自该模型的大量初步数据有力地表明，与现有的 在体内，弓形虫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中枢神经系统细胞。这项建议的目标是确定神经元-弓形虫界面如何驱动中枢神经系统感染的建立和结果。为此，我们将使用我们的新小鼠模型与两种基因分化的弓形虫菌株相结合，这两种菌株会导致不同的中枢神经系统结果，我们已经设计好注射Cre。具体地说，使用我们开发的切割边缘中枢神经系统成像技术，我们将确定这些弓形虫菌株是否通过感染不同的中枢神经系统区域或增选不同的神经元亚型而导致不同的中枢神经系统结果(目标1)。此外，为了确定这些菌株特异性的中枢神经系统效应是否继发于神经元的差异操作，我们将分离并转录描述这些弓形虫注射的神经元(目标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中枢神经系统疾病提供第一个机械性见解。最终，这些见解将为我们提供新的途径，开发针对中枢神经系统的、甚至针对菌株的治疗方法，以改善中枢神经系统疾病的结果。

 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.