Genetic dissection of the Toxoplasma gondii rhoptry kinome

描述（由申请人提供）： 弓形虫是艾滋病患者重要的机会性感染。迫切需要改进策略和方法，以更有效地预防和治疗艾滋病复发感染。原发感染后，一个慢性终身T。在CNS/脑中建立了以存在含有缓慢或非复制性缓殖子寄生虫形式的持续性包囊为特征的弓形虫感染。在免疫抑制过程中由于囊肿破裂而引起的再活化感染导致艾滋病中难以治疗且危及生命的弓形虫脑炎。目前，没有疫苗被批准用于人类以预防感染，并且用于急性感染的药物治疗是次优的。根除预先存在的囊肿和慢性感染是控制/预防艾滋病感染的一种很好的方法。然而，目前没有有效的治疗方法可以根除预先存在的囊肿和慢性感染。在针对囊肿阶段方面缺乏进展可归因于我们对囊肿发展和慢性感染维持的寄生虫生物学知识的重大差距。最近的进展，在了解复杂的宿主-寄生虫相互作用显示，许多棒状球（ROP）蛋白似乎是中央球员在宿主细胞操作，由于它们的分泌到宿主细胞的入侵和随后的本地化到宿主细胞的胞质溶胶/核或其密切的协会与寄生虫空泡。通过这些分泌的ROP蛋白的宿主细胞操纵可能对体内急性和慢性感染所必需的寄生虫生物学的成功至关重要。寄生虫空泡似乎是速殖子复制的关键，也是慢性感染特征的囊壁和组织囊发育的中心。我们假设，具有与激酶同源性的~ 34种分泌型ROP蛋白质的家族（“ROP激酶组”）是作为破坏毒力、囊肿发育或慢性感染的潜在靶标的突出候选者。在这里，我们建议删除II型T中分泌型ROP激酶组的每个预测基因成员。弓形虫，然后确定急性毒力，早期包囊发育或慢性感染中的任何缺陷。使用定向功能遗传学方法，这项工作将确定参与建立或维持体内慢性感染所需的关键功能的分泌型ROP激酶组的特定成员。该项目将鉴定和验证特定的分泌型ROP激酶作为预防急性或慢性感染的关键靶点。该项目还将开发必要的工具和方法，用于未来开展更大规模的高通量敲除和II型T的功能基因组项目。刚地。因此，这一创新项目通过推进II型生物学解剖的遗传模型，揭示宿主-寄生虫相互作用的新方面，以及揭示预防或根除慢性感染的新靶点，具有很高的整体影响力。 公共卫生相关性： 该项目将鉴定弓形虫建立和维持慢性感染能力所必需的专门寄生虫蛋白。目前没有预防感染的疫苗，也没有根除慢性感染的药物治疗。通过对一个专门的寄生虫蛋白家族的功能分析，我们期望确定预防或根除慢性感染的新靶点。在这个项目中获得的信息有助于揭示寄生虫如何操纵哺乳动物细胞和哺乳动物宿主的基本知识，从而揭示根除人类重要寄生虫的新方法。

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. Following primary infection, a chronic life-long T. gondii infection characterized by the presence of persisting cysts containing slowly or nonreplicating bradyzoite parasite forms is established in CNS/brain. Reactivated infection due to cyst rupture during immune suppression causes a difficult to treat and life-threatening Toxoplasmic encephalitis in AIDS. Currently, no vaccine is approved for use in humans to prevent infection and drug treatments for acute infection are suboptimal. Eradicating pre-existing cysts and chronic infection is an excellent approach to control/prevent infection in AIDS. However, no current treatment is effective at eradicating pre-existing cysts and chronic infection. Lack of progress in targeting cyst stages can be attributed to major gaps in our knowledge of parasite biology underlying cyst development and maintenance of chronic infection. Recent progress in understanding the complex host-parasite interaction has revealed that many rhoptry bulb (ROP) proteins appear to be central players in host cell manipulation due to their secretion into the host cell at invasion and their subsequent localization to host cell cytosol/nucleus or their intimate association with the parasitophorous vacuole. Host cell manipulation by these secreted ROP proteins is likely to be critical to the success of parasite biology necessary for acute and chronic infection in vivo. The parasitophorous vacuole appears to be critical for replication of tachyzoites as well as being central to the development of the cyst wall and tissue cyst that characterize chronic infection. We hypothesize that a family of ~ 34 secreted ROP proteins possessing homology to kinases (the "ROP kinome") are outstanding candidates as potential targets to disrupt virulence, cyst development, or chronic infection. Here, we propose to delete each predicted gene member of the secreted ROP kinome in type II T. gondii, and to then ascertain any defect in acute virulence, in early cyst development, or in chronic infection. Using a directed functional genetic approach, this work will identify specific members of the secreted ROP kinome that participate in critical functions required for establishing or maintaining chronic infection in vivo. This project will identify and validate specific secreted ROP kinases as key targets for preventing acute or chronic infection. This project will also develop essential tools and methods required for the future undertaking of larger scale higher throughput knockout and functional genomic projects in type II T. gondii. Consequently, this innovative project has high overall impact by advancing genetic models for dissection of type II biology, by revealing new aspects of host-parasite interaction, and by exposing new targets to prevent or eradicate chronic infection. PUBLIC HEALTH RELEVANCE: This project will identify specialized parasite proteins that are essential for the ability of Toxoplasma gondii to establish and maintain a chronic infection. No vaccine is currently available to prevent infection and no drug treatment is available that can eradicate chronic infection. By functional analysis of a family of specialized parasite proteins we expect to identify new targets to prevent or eradicate chronic infection. The information gained in this project helps to uncovering fundamental knowledge of how a parasite manipulates mammalian cells and the mammalian host, and will thus uncover new approaches to eradicate a significant parasite of humans.