Employing Toxoplasma gondii virulence mutants to examine protective immunity

描述（由申请人提供）：弓形虫是一种专性细胞内寄生虫，感染几乎所有哺乳动物和鸟类有核细胞。免疫功能正常的个体感染弓形虫通常无症状；然而，弓形体病可能对胎儿和免疫功能低下的个体（例如艾滋病患者和器官移植受者）产生严重影响。毒力突变体已在带有特征标记的诱变文库中产生并鉴定。拟议的研究将鉴定文库中毒性较低且无法引起典型急性感染的突变体。这些突变体将用于勾勒出保护性免疫特征，并深入了解可能的药物靶点，以限制最有严重弓形体病风险的个体中弓形虫的感染。将补充体内筛选中鉴定出的弓形虫毒力突变体，以确认引起突变表型的基因。将确定毒力基因的蛋白质产物的表达水平和位置。将构建毒力基因的敲除突变体并用于在注射致死剂量的弓形虫之前对小鼠进行免疫。保护性免疫的效率将通过 LD50 的变化和慢性感染阶段每个大脑的囊肿数量来量化。将通过测量小鼠中诱导的细胞因子和感染期间寄生虫的传播来检查保护概况。弓形虫是一种寄生病原体，可导致胎儿和免疫功能低下的个体（包括艾滋病患者和移植受者）出现严重且危及生命的疾病。拟议的研究将利用急性毒力突变体来检查保护性免疫，并深入了解可能的药物靶标，以保护这些个体免受弓形虫的严重感染。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite that infects nearly all mammalian and avian nucleated cells. Infection of immunocompetent individuals with T. gondii is generally asymptomatic; however, toxoplasmosis can have severe implications in fetuses and immunocompromised individuals, such as AIDS patients and organ transplant recipients. Virulence mutants have been generated and identified in a signature-tagged mutagenesis library. The proposed research will characterize mutants from the library that are less virulent and unable to cause a typical acute infection. The mutants will be used to outline a protective immunity profile and provide insight into possible drug targets to limit infection of T. gondii in individuals most at risk for severe toxoplasmosis. T. gondii virulence mutants identified in an in vivo screen will be complemented to confirm the gene causing the mutant phenotype. The expression level and location of the protein product of the virulence gene will be determined. A knockout mutant of the virulence genes will be constructed and used to immunize mice before injection of a lethal dose of T. gondii. Efficiency of protective immunity will be quantified by change in LD50 and the number of cysts per brain during the chronic phase of infection. The protective profile will be examined through measurements of cytokines induced in mice and dissemination of the parasites during infection. Toxoplasma gondii is a parasitic pathogen that can cause severe and life-threatening illnesses in fetuses and immunocompromised individuals, including AIDS patients and transplant recipients. The proposed research will utilize acute virulence mutants to examine protective immunity and provide insight into possible drug targets to protect these individuals from serious infection with T. gondii.