Characterizing Toxoplasma gondii encystation mutants

项目简介：刚地弓形虫（Toxoplasma gondii）是世界范围内危害人类和牲畜的严重病原体。弓形虫可引起胎儿发育异常或发育中的胎儿死亡，免疫功能低下患者可引起脑炎。该项目的最终目标是开发一种能够预防弓形虫感染的疫苗。任何弓形虫活疫苗的一个关键问题是，该菌株不能形成持久的囊肿，这是慢性感染的标志。从快速生长阶段到成囊阶段的转化对寄主内的持久性至关重要，尽管人们对这种相互转化知之甚少。在这个项目中，参与囊肿形成的基因将被表征，并将确定一个环孢突变体作为候选疫苗株。在体外和体内筛选了刚地弓形虫插入突变体库，以寻找内嵌缺陷。从这些屏幕中识别出的九个突变体是本应用程序的重点。这些突变体将在体内定量分析囊肿形成。除一个突变体外，所有突变体的诱变质粒的插入位点都已确定。将确定该剩余突变体的插入位点，并通过北方杂交确定中断的基因。突变体将通过被破坏基因的完整拷贝的补充而得到补充，这表明这些基因座参与了囊肿的形成。将测试选定的突变体在小鼠中维持持续感染的能力。针对已鉴定的囊肿发育因子产生的抗血清将用于表达和定位研究，以表征这些蛋白。不能在小鼠体内维持囊肿的菌株将被检查其保护小鼠免受致命的弓形虫感染的能力。相关性：我们的研究重点是确定生长缓慢、持久形式的弓形虫发展的重要因素，并产生一种无法过渡到这种形式或长期留在宿主体内的寄生虫菌株。这种菌株将作为疫苗进行测试，以帮助免疫系统清除感染寄生虫，然后让它们转化为持久的形式。

DESCRIPTION (provided by applicant): Project Summary: The parasite Toxoplasma gondii is a serious pathogen of humans and livestock worldwide. T. gondii causes abnormal fetal development or death in a developing fetus, and encephalitis in immunocompromised patients. The ultimate goal of this project is to develop a vaccine able to protect against T. gondii infection. A key concern with any live T. gondii vaccine is that the strain be unable to form persistent cysts, the hallmark of chronic infection. Conversion from the fast growing stage to the encysted stage is critical for persistence within the host, though little is known about this interconversion. In this project, genes involved in cyst formation will be characterized, and an encystation mutant will be identified as a vaccine strain candidate. A library of T. gondii insertional mutants was screened in vitro and in vivo for encystation defects. Nine mutants identified from these screens are the focus of this application. These mutants will be analyzed quantitatively for cyst formation in vivo. The insertion site of the mutagenizing plasmid has been identified for all but one mutant. The insertion site will be determined for this remaining mutant, and the disrupted genes will be confirmed by northern hybridization. Mutants will be complemented by resupply of intact copies of the disrupted genes, demonstrating that these loci are involved in cyst formation. Select mutants will be tested for their ability to maintain a persistent infection in mice. Antisera generated against the identified cyst development factors will be used in expression and localization studies to characterize these proteins. Strains unable to maintain cysts in mice will be checked for their ability to protect mice from lethal infection of T. gondii. Relevance: Our research focuses on identifying factors important in the development of the slow-growing, persistent form of T. gondii, and generating a strain of the parasite unable to transition to this form or remain in the host long-term. This strain will be tested as a vaccine to help the immune system clear infecting parasites before allowing them to convert to the persistent form.