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重要机会性感染。迫切需要改进战略和方法，以更有效地预防和治疗艾滋病复发感染。由于在体外培养和克隆，转化效率，丰富的建立遗传工具，和强大的感染小鼠模型的可用性，弓形虫越来越多地被认为是一个模型apicomplexan寄生虫了解细胞内寄生和宿主免疫反应的生物学。本实验室已开发出一种尿嘧啶营养缺陷型（cps1 - 1）突变株。弓形虫（RH背景），被证明是一个有价值的模式生物，了解关键的宿主-寄生虫相互作用。cps1 - 1突变体侵入宿主细胞，但在体外或体内缺乏尿嘧啶时不能复制。尿嘧啶营养缺陷型cps1 - 1的速殖子可以在体外培养，并且在腹膜内（i.p.）接种到小鼠中引发有效Th1应答，其赋予有效的长期保护以免受I型强毒株RH的致死攻击感染。我们最近的研究结果表明，cps1 - 1的ip.e.给药后，令人惊讶的有效的长期保护性免疫力，从致命的原发性和慢性感染后，经口挑战II型ME49囊肿。最常见的是，人体接触和传播弓形虫的自然途径是通过口服组织包囊。从囊肿释放的缓殖子在肠粘膜中发起原发性感染，最终导致慢性脑囊肿的建立，该慢性脑囊肿随后可引起AIDS中的弓形虫脑炎。T.肠粘膜中的弓形虫感染可能在引发导致免疫保护的先天性和适应性免疫应答中起关键作用。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陷型，目标是开发可以通过天然口服途径递送的减毒活囊肿形成突变体。本项目中开发的菌株应提供解剖肠道粘膜早期感染中关键宿主-寄生虫相互作用所需的宝贵工具，并将作为研究宿主反应、寄生虫发育、贩运、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，本项目中开发的模式生物可能为实验性解决囊肿形成、囊肿维持和囊肿复发提供宝贵的工具。本研究旨在开发和验证具有毒力和发育调控特性的弓形虫模式生物株。这些模式生物预计将提供显着改善的能力，进行关键的调查宿主反应，寄生虫的发展，和宿主-寄生虫生物学，因为这些领域涉及到新的概念，有效的疫苗接种，以预防或治疗脑囊肿。因此，该研究项目将加速发现新的疫苗和治疗艾滋病中毁灭性的机会性寄生虫感染的方法。

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.