Regulated virulence and cyst maturation in Toxoplasma gondii

描述（申请人提供）：刚地弓形虫是艾滋病患者重要的机会性感染。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性感染。由于体外培养和克隆的方便性、转化的效率、已建立的遗传工具的丰丰性以及强大的小鼠感染模型的可用性，弓形虫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一种模式顶复合体寄生虫。我们的实验室已经开发出一种高度减毒的弓形虫（RH背景）尿嘧啶营养不良（cps1-1）突变体，它被证明是一种有价值的模式生物，用于理解宿主-寄生虫的关键相互作用。cps1-1突变体侵入宿主细胞，但在没有尿嘧啶的情况下，在体外或体内都不能复制。尿嘧啶营养不良细胞cps1-1的速殖子可以在体外培养，并通过腹腔注射（i.p.）接种到小鼠体内，引起有效的Th1反应，从而有效地长期保护小鼠免受强毒I型RH菌株的致命攻击感染。我们最近的研究结果表明，cps1-1给予ipp可在II型ME49囊肿经口攻击后引起致命的原发性和慢性感染的长期保护性免疫。最常见的是，人类接触和传播弓形虫的自然途径是通过口服摄入组织囊肿。从囊肿中释放的慢殖子在肠道黏膜中引发原发性感染，最终导致慢性脑囊肿的形成，随后可引起艾滋病的弓形虫脑炎。弓形虫感染肠道黏膜时，早期宿主-寄生虫相互作用可能是引发先天和适应性免疫反应的关键，从而导致免疫保护。我们建议在I型和II型菌株中开发受tet调节的尿嘧啶缺陷增生，目的是开发可通过自然经口途径传递的减毒活囊形成突变体。该项目开发的菌株将为解剖肠道黏膜早期感染中关键宿主-寄生虫相互作用提供必要的有价值的工具，也将成为研究宿主反应、寄生虫发育、运输、囊肿生物学、免疫和疫苗的创新模型。与艾滋病的弓形虫脑炎感染有关，本项目中开发的模式生物可能为实验解决囊肿形成，囊肿维持和囊肿复发提供宝贵的工具。本研究项目将开发和验证具有可调控毒力和发育特性的刚地弓形虫模型生物菌株。这些模式生物有望为宿主反应、寄生虫发育和宿主-寄生虫生物学的关键研究提供重大改进，因为这些领域涉及有效疫苗接种以预防或治疗脑囊肿的新概念。因此，这一研究项目将加速发现新的疫苗和治疗艾滋病中的破坏性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.