The Toxoplasma basal complex in cell division

细胞分裂中的弓形虫基础复合体

• 批准号: 10328552
• 负责人: Marc-Jan Gubbels
• 金额: $ 37.53万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-10 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10328552
• 关键词: Actins; Adherence; Adhesions; Affect; Alveolar; Apical; Binding; Biological; Biotinylation; C-terminal; Cell Division Process; Cell division; Cells; Centrosome; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Congenital Abnormality; Cytoskeleton; Data; Data Set; Daughter; Dissection; Drug Targeting; Encephalitis; Enzymes; Family; Genes; Genetic; Grant; Hypersensitivity; In Vitro; Intermediate Filament Proteins; Kinetics; Knock-out; Libraries; Lytic Phase; Maps; Membrane; Microtubule-Associated Proteins; Microtubules; Modeling; Mothers; Motor; Myosin ATPase; N-terminal; Opportunistic Infections; PAWR protein; Parasites; Parents; Pathology; Pharmaceutical Preparations; Phenocopy; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Population; Process; Protein C; Proteins; Proteomics; Recombinants; Regulation; Resistance; Role; Running; Scaffolding Protein; Signal Transduction; Solid; Structure; Testing; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vesicle; Xenopus; axon guidance; base; candidate validation; constriction; daughter cell; depolymerization; factor EF-P; fitness; foodborne infection; genome-wide; inorganic phosphate; insight; knock-down; membrane skeleton; microtubule-associated protein 1B; mutant; new therapeutic target; overexpression; prevent; protein complex; recruit; scaffold; screening; spatiotemporal

Summary Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing severe opportunistic infections. Current drugs are prone to induce hypersensitivity, especially upon long-term use. Under this proposal the unique cell division process will be interrogated to identify putative new drug targets. Toxoplasma divides by a distinct internal budding process whereby two daughter parasites are assembled within a mother cell. The cortical membrane skeleton composed of flattened alveolar vesicles supported by an epiplastin protein network and 22 subpellicular microtubules (MTs) is nucleated on the centrosomes and assembles in an apical to basal direction. In the second half of division the posterior end of the daughter buds (i.e. the basal complex or BC) starts to taper driven by Myosin J (MyoJ). Absence of MyoJ only modestly impact parasite viability, even while it leaves the BC somewhat unconstricted, fitting classic data on cell division resistance to actin depolymerizing agents. However, preventing assembly of the BC altogether by depleting or overexpressing the BC scaffolding protein MORN1 results in parasites with fraying MTs unable to complete cell division and has dramatic impact on viability. To unravel this intriguing process, under an R21 grant the BC was proteomically dissected through proximity dependent biotinylation (BioID) on 8 BC components. This revealed 4-5 different protein complexes aligning with the ultrastructure. Two key observations are further pursued under this proposal: 1. A putative MT Associated Protein, MAP1B-L1, appears to assemble on the (+)-ends of the subpellicular MTs and is essential for BC assembly and parasite viability; 2. Several kinases and phosphatases identified indicate the BC is regulated by differential phosphorylation. Under Aim 1 MAP1B-L1 and another critical BC MAP dubbed MAP1B- L2 will be tested for MT binding capacity by generating deletion mutants in the parasite, in vitro using the identified MT binding domains, and by exogenous expression in the Xenopus leavis axon guidance model as relevant to related MAPs. Under Aim 2 we will pursue four additional candidates identified in the BioID approach with a likely essential function, which are all hypothetical proteins narrowly conserved in internally budding parasites and harbor putative adhesion domains. In addition, we will apply fast acting TurboID on BC components transiently associating with the assembling BC like MAP1B-L1 as these were likely undersampled in the current dataset, yet define the essential step of the BC in cell division. Under Aim 3 we will subject 2 kinases and 1 phosphatase to synthetic lethality screening using the genome wide CRISRP/Cas9 library. Preliminary data of the first kinase tested already demonstrates experimental feasibility and revealed interesting new insights. Combining the proteomic and genetic data sets is expected to provide a solid basis to assemble the wiring diagram of the BC. In the current working model the BC is first assembled on the MT (+)-ends, followed by recruitment of adhesion proteins to keep the MT-ends together. Overall, this is expected to deliver exciting new insights into internal budding, how it differs from schizogony, and could highlight new drug targets.

总结 弓形虫是一种专性细胞内顶复体寄生虫，可引起严重的机会性感染。 目前的药物容易诱发超敏反应，特别是在长期使用时。根据这一提议， 独特的细胞分裂过程将被询问，以确定推定的新药靶点。弓形虫分为 两个子寄生虫在母细胞内组装的独特的内部出芽过程。的 皮质膜骨架，由扁平的肺泡囊泡组成，由表质体蛋白质网络支撑 22个膜下微管（subpellicular microtubules，MT）在中心体上成核，并以顶端到基部的方式组装 方向在分裂的后半部分，子芽的后端（即基部复合体或BC） 肌球蛋白J（MyoJ）开始逐渐减少。缺乏MyoJ仅适度影响寄生虫的生存能力，即使它 使BC有点不受约束，适合细胞分裂抵抗肌动蛋白解聚的经典数据 剂.然而，通过耗尽或过度挤压BC支架来完全阻止BC的组装， 蛋白MORN 1导致带有磨损的MT的寄生虫无法完成细胞分裂， 关于生存能力。为了解开这个有趣的过程，在R21的资助下，BC被蛋白质组学解剖， 在8个BC组分上的邻近依赖性生物素化（BioID）。这揭示了4-5种不同的蛋白质复合物 与超微结构一致。本建议进一步提出两项关键意见：1.假定的MT 相关蛋白MAP 1B-L1似乎组装在膜下MT的（+）-末端，并且是必需的 对于BC组装和寄生虫活力; 2.鉴定的几种激酶和磷酸酶表明BC是 由差异磷酸化调节。在目标1 MAP 1B-L1和另一个关键的BC MAP（称为MAP 1B-L1）下， 将通过在寄生虫中产生缺失突变体，在体外使用 鉴定的MT结合结构域，并通过在非洲爪蟾轴突指导模型中的外源表达， 与相关的地图。在目标2下，我们将继续研究BioID方法中确定的另外四种候选药物 具有可能的基本功能，这些都是假设的蛋白质，在内部出芽中保守， 寄生虫和港口推定的粘附结构域。此外，我们将在BC组件上应用快速作用的TurboID 与组装BC（如MAP 1B-L1）暂时关联，因为这些在当前可能采样不足 数据集，但定义了细胞分裂中BC的基本步骤。在目标3下，我们将研究2种激酶和1种 使用全基因组CRISRP/Cas9文库进行磷酸酶对合成致死性筛选。初步数据 测试的第一种激酶已经证明了实验的可行性，并揭示了有趣的新见解。 结合蛋白质组学和遗传学数据集有望为组装连接提供坚实的基础 图为BC。在目前的工作模型中，BC首先组装在MT（+）端上，然后是 募集粘附蛋白以保持MT末端在一起。总的来说，这有望带来令人兴奋的新 深入了解内部萌芽，它如何不同于胚胎发育，并可能突出新的药物靶点。