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。迫切需要改进战略和方法，以便更有效地预防和治疗艾滋病复发感染。在初次感染后，在中枢神经系统/脑中建立了一种慢性终生弓形虫感染，其特征是存在含有缓慢或不复制的慢速殖子寄生虫形式的持续包囊。在免疫抑制过程中，由于包囊破裂而引起的再激活感染导致艾滋病中的弓形体脑炎难以治疗，并危及生命。目前，没有疫苗被批准用于人类预防感染，急性感染的药物治疗也不是最理想的。根除先前存在的包囊和慢性感染是控制/预防艾滋病感染的一个很好的办法。然而，目前没有一种治疗方法能有效根除先前存在的包囊和慢性感染。在以囊肿期为靶点方面缺乏进展可归因于我们对包囊发育和维持慢性感染的寄生虫生物学知识的主要空白。最近对宿主-寄生虫相互作用的研究进展表明，许多棒状病毒球蛋白(ROP)似乎是宿主细胞操纵的中心角色，因为它们在入侵时分泌到宿主细胞中，随后定位到宿主细胞的胞浆/细胞核或与寄生虫的空泡密切相关。这些分泌的ROP蛋白对宿主细胞的操纵很可能是体内急、慢性感染所必需的寄生虫生物学成功的关键。寄生空泡似乎是速殖子复制的关键，也是囊壁和组织囊形成的中心，囊壁和组织囊是慢性感染的特征。我们假设~34个与蛋白激酶具有同源性的分泌的ROP蛋白家族(“ROP激动组”)是破坏毒力、包囊发育或慢性感染的潜在靶点。在这里，我们建议删除II型弓形虫分泌的ROP激动组的每个预测基因成员，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，这项工作将识别分泌的ROP激动组的特定成员，这些成员参与建立或维持体内慢性感染所需的关键功能。该项目将确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目标。该项目还将开发未来开展更大规模、更高产量的敲除和II型弓形虫功能基因组项目所需的必要工具和方法。因此，这一创新项目通过推进解剖II型生物的遗传模型、揭示宿主与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目标，具有很高的整体影响。 公共卫生相关性： 该项目将确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。目前还没有预防感染的疫苗，也没有可以根除慢性感染的药物治疗。通过对一系列特殊寄生虫蛋白的功能分析，我们希望确定预防或根除慢性感染的新靶点。从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，从而揭示根除人类一种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.