Regulated virulence and cyst maturation in Toxoplasma gondii

描述(申请人提供)：弓形虫是艾滋病患者的一种重要的机会性感染。迫切需要改进战略和方法，以便更有效地预防和治疗艾滋病复发感染。由于弓形虫易于体外培养和克隆，转化效率高，成熟的遗传工具丰富，以及可获得强大的小鼠感染模型，因此越来越被认为是了解细胞内寄生和宿主免疫反应生物学的顶复合体模式寄生虫。我们实验室已经开发出弓形虫高度减毒的尿嘧啶缺陷型(cps1-1)突变体(RH背景)，它被证明是理解关键宿主-寄生虫相互作用的一个有价值的模式生物。Cps1-1突变体侵入宿主细胞，但在没有尿嘧啶的情况下不会在体外或体内复制。尿嘧啶辅助体cps1-1的速殖子可以在体外培养，也可以在腹膜内培养(I.P.)接种到小鼠身上会引起强大的Th1反应，从而对来自I型毒株RH的致死性攻击感染提供有效的长期保护。我们最近的研究结果表明，cps1-1通过ip给药。在经口攻击II型ME49型包囊后，对致死性原发和慢性感染产生令人惊讶的有效长期保护性免疫。最常见的是，弓形虫接触和传播给人类的自然途径是通过口服组织包囊。从包囊中释放出的缓殖子在肠道粘膜中发起原发感染，最终导致慢性脑囊肿的建立，随后可能会导致艾滋病的弓形体脑炎。弓形虫感染早期肠道粘膜中宿主和寄生虫的相互作用可能在激发先天和获得性免疫反应从而导致免疫保护方面起关键作用。我们建议在I型和II型菌株中开发tet调节的尿嘧啶营养缺乏症，目标是开发可以通过自然经口途径传递的减毒活包囊形成突变体。该项目中开发的菌株将为剖析肠道粘膜早期感染中关键的宿主-寄生虫相互作用提供必要的有价值的工具，并将作为研究宿主反应、寄生虫的发展、运输、包囊生物学、免疫和疫苗的创新模型。与艾滋病中的弓形虫脑炎感染相关，该项目中开发的模式生物可能为实验解决包囊形成、包囊维持和包囊复发提供宝贵的工具。本研究项目将开发和验证具有调节毒力和发育特性的弓形虫模式生物菌株。这些模式生物有望显著提高对宿主反应、寄生虫发育和宿主-寄生虫生物学进行关键调查的能力，因为这些领域与有效接种疫苗以预防或治疗脑囊肿的新概念有关。因此，这项研究项目将加速发现新的疫苗和治疗方法，以应对艾滋病中毁灭性的机会性寄生虫感染。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an important opportunistic infection of AIDS patients. Improved strategies and approaches are urgently needed to more effectively prevent and treat recurrent infections in AIDS. Due to the ease of in vitro culture and cloning, efficiency of transformation, abundance of established genetic tools, and availability of robust murine models of infection, Toxoplasma gondii is increasingly recognized as a model apicomplexan parasite for understanding biology of intracellular parasitism and host immune response. Our laboratory has developed a highly attenuated uracil auxotroph (cps1-1) mutant of T. gondii (RH background) that is proving to be a valuable model organism for understanding critical host-parasite interactions. The cps1-1 mutant invades host cells, but does not replicate in vitro or in vivo in the absence of uracil. Tachyzoites of the uracil auxotroph cps1-1 can be cultured in vitro and following intraperitoneal (i.p.) inoculation into mice elicit potent Th1 responses that confer effective long-term protection from a lethal challenge infection from virulent type I strain RH. Our recent results demonstrate that cps1-1 administered i.p. elicits surprisingly effective long-term protective immunity from lethal primary and chronic infections after peroral challenge with type II ME49 cysts. Most commonly, the natural route of exposure and transmission of Toxoplasma to humans is via oral ingestion of tissue cysts. Bradyzoites released from cysts launch a primary infection in gut mucosa that ultimately leads to the establishment of chronic brain cysts that can later cause Toxoplasmic encephalitis in AIDS. The early host-parasite interactions during T. gondii infection in the gut mucosa are likely to be critical in eliciting innate and adaptive immune responses that lead to immune protection. We propose to develop tet regulated uracil auxotrophy in type I and type II strains with a goal to develop live-attenuated cyst-forming mutants that can be delivered by the natural peroral route. The strains developed in this project should provide valuable tools necessary for the dissection of key host- parasite interactions in early infection in gut mucosa and will also serve as innovative models for studying host response, parasite development, trafficking, cyst biology, immunity, and vaccines. Relevant to Toxoplasmic encephalitis infections in AIDS, the model organisms developed in this project are likely to provide invaluable tools for experimentally addressing cyst formation, cyst maintenance, and cyst recrudescence. This research project will develop and validate model organism strains of Toxoplasma gondii with the property of regulated virulence and development. These model organisms are expected to provide significant improvements in the ability to perform critical investigations into host response, parasite development, and host-parasite biology as these areas relate to new concepts for effective vaccination to prevent or treat brain cysts. Consequently, this research project will accelerate the discovery of new vaccines and treatments for devastating opportunistic parasite infections in AIDS.