Molecular mechanisms of pathogenesis in Toxoplasma

描述（由申请人提供）： 我的长期研究目标是研究细胞内病原体弓形虫的毒力机制，并确定寄生虫株类型在决定疾病结局中的作用。T.刚地虫是顶复门的成员，该门包括其它人类寄生虫，包括疟疾和隐孢子虫病的病原体。在人类中，弓形虫是一种机会致病菌，在发育中的胎儿和免疫功能低下的个体中感染特别严重。虽然已知不同的弓形虫株在小鼠（可能还有人类）中引起不同的疾病，但直到最近才确定了负责的寄生虫蛋白质。我们和其他人最近发现了一种寄生虫来源的分泌蛋白激酶（棒状蛋白18; ROP 18），它使小鼠中无毒力弓形虫的致死率增加了4个log以上。虽然ROP 18对小鼠毒力的影响是明确的，但其作用机制知之甚少，本项目的目标是确定ROP 18对毒力影响的分子和细胞机制。要做到这一点，两个互补的方法进行说明。在目的1中，我将鉴定在体内腹膜内感染期间由ROP 18操纵的细胞和分子信号传导事件。我将确定转录，细胞因子分泌，和免疫细胞类型的招聘与菌株感染的ROP 18表达毒力和无毒力形式的差异，并与疾病的进展，使用体内生物发光成像。在目标2中，我将开发针对ROP 18的激酶结构域的小分子探针，以允许在体内直接操纵ROP 18激酶活性并鉴定ROP 18的生理底物。这些方法应该对ROP 18的分子靶点产生新的见解，这些靶点与其对毒力的影响有关，并增加我们对一般毒力机制的理解。此外，它们可能导致分子标记物的发展，这些标记物将允许预测人类弓形虫感染的严重程度，从而可以相应地调整治疗方案。相关性（参见说明）：弓形虫病是一种在艾滋病患者和发育中的胎儿中可能非常严重的疾病，并且似乎感染的严重程度至少部分取决于弓形虫菌株类型。该项目的目标是了解为什么某些弓形虫菌株比其他菌株引起更严重的疾病。这将提高我们诊断更具侵袭性感染的能力，并更有效地开出治疗处方。

DESCRIPTION (provided by applicant): My long-term research goal is to study the mechanisms of virulence in the intracellular pathogen Toxoplasma gondii, and determine the role of parasite strain type in determining disease outcome. T. gondii is a member of the phylum Apicomplexa which includes other human parasites, including the causative agents of malaria and cryptosporidiosis. In humans Toxoplasma is an opportunistic pathogen, and infections are particularly severe in the developing fetus and in individuals who have become immunocompromised. While it was known that different strains of Toxoplasma caused different disease in mice (and possibly humans), until recently the parasite proteins responsible had not been identified. We, and others, have recently identified a parasite-derived secreted protein kinase (rhoptry protein 18; ROP18) that increases the lethality of avirulent Toxoplasma in mice by over 4 logs. While the effect of ROP18 on virulence in mice is clear, its mechanism of action is poorly understood, and it is the goal of this project to determine the molecular and cellular mechanisms for the impact of ROP18 on virulence. To do this two complementary approaches are described. In Aim 1 I will identify the cellular and molecular signaling events that are manipulated by ROP18 during intraperitoneal infection in vivo. I will identify transcriptional, cytokine secretion, and immune cell type recruitment differences in mice infected with strains expressing virulent and avirulent forms of ROP18, and correlate them with disease progression using in vivo bioluminescence imaging. In Aim 2 I will develop small-molecule probes for the kinase domain of ROP18 to allow for the direct manipulation of ROP18 kinase activity in vivo and for the identification of the physiological substrates of ROP18. These approaches should yield new insights into the molecular targets of ROP18 that are relevant to its effects on virulence, and increase our understanding of the mechanisms of virulence in general. Moreover they may result in the development of molecular markers that would allow the severity of human Toxoplasma infections to be predicted so that treatment regimens could be tailored accordingly. RELEVANCE (See instructions): Toxoplasmosis is a disease that can be very severe in AIDS patients and the developing fetus, and it appears that the severity of infection is at least partially dependent on Toxoplasma strain type. The goal of this project is to understand why some strains of Toxoplasma cause more severe disease than others. This will improve our ability to diagnose more aggressive infections and prescribe treatments more effectively.