Employing Toxoplasma gondii virulence mutants to examine protective immunity

描述（由申请人提供）：刚地弓形虫是一种专性细胞内寄生虫，几乎感染所有哺乳动物和鸟类的有核细胞。具有免疫功能的个体感染弓形虫通常是无症状的；然而，弓形虫病可能对胎儿和免疫功能低下的个体（如艾滋病患者和器官移植接受者）产生严重影响。毒力突变体已经在一个签名标记的诱变库中产生和鉴定。拟议的研究将从库中描述毒性较低且不能引起典型急性感染的突变体。这些突变体将用于概述保护性免疫概况，并为可能的药物靶点提供见解，以限制严重弓形虫病最危险个体的弓形虫感染。将补充在体内筛选中鉴定的弓形虫毒力突变体，以确认引起突变表型的基因。毒力基因蛋白产物的表达水平和位置将被确定。将构建毒力基因的敲除突变体，并在注射致死剂量的弓形虫之前用于免疫小鼠。在感染的慢性期，将通过LD50的变化和每个脑的囊肿数量来量化保护性免疫的效率。将通过测量小鼠体内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。刚地弓形虫是一种寄生病原体，可在胎儿和免疫功能低下的个体（包括艾滋病患者和移植接受者）中引起严重和危及生命的疾病。拟议的研究将利用急性毒力突变体来检查保护性免疫，并提供可能的药物靶点，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.