Elucidation of TgPKG kinase substrates required for Toxoplasma motility

阐明弓形虫运动所需的 TgPKG 激酶底物

• 批准号: 10674317
• 负责人: Kevin Michael Brown
• 金额: $ 17.75万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10674317
• 关键词: Acute; Amino Acids; Auxins; Biotin; Cell Culture Techniques; Cell membrane; Cells; Chemicals; Chronic; Co-Immunoprecipitations; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease; Drug Targeting; Epitopes; Gene Expression; Genes; Genetic; Guanosine Triphosphate; Guanylate Cyclase; Human; Immunity; Infection; Infection prevention; Kinetics; Label; Life; Liquid Chromatography; Modeling; Modernization; Oklahoma; Organism; Paralysed; Parasites; Pathogenesis; Peptides; Permeability; Pharmaceutical Preparations; Phenocopy; Phosphopeptides; Phosphorylation; Phosphotransferases; Plasmodium; Positron-Emission Tomography; Proteins; Proteomics; Pyrimethamine; Stable Isotope Labeling; Sulfadiazine; Testing; Tissues; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Tumor stage; United States National Institutes of Health; Vaccines; Virulence; analog; cell motility; differential expression; drug development; experimental study; extracellular; insight; knock-down; microbial; phosphoproteomics; tandem mass spectrometry; transcription factor; transcriptome sequencing; transcriptomics

Specific Aims Toxoplasma gondii persists in 25-30% of humans worldwide because there are no vaccines to prevent infections and current therapies are non-curative (1). The first-line therapy for toxoplasmosis consists of pyrimethamine and sulfadiazine, which inhibit intracellular parasite replication but are highly toxic and unable to eliminate the dormant tissue-encysted stage of T. gondii (2). Thus, there is an urgent need for new non-toxic drugs that block T. gondii infectivity in both acute (tachyzoite) and chronic (bradyzoite) life stages. Cyclic guanosine monophosphate (cGMP) has emerged as a master regulator of T. gondii infectivity by coordinating parasite motility (3, 4). Previously, we determined that a T. gondii guanylate cyclase (TgGC) synthesizes cGMP from GTP to stimulate motility for entry and exit of host cells (5). Conditional depletion of TgGC paralyzed T. gondii, rendering parasites incapable of establishing infections and causing disease. Additionally, we determined that a cGMP-dependent protein kinase (TgPKG) at the plasma membrane acts as the central effector of cGMP in T. gondii in both tachyzoites (6) and bradyzoites (7). Chemical inhibition or conditional knockdown of TgPKG phenocopied loss of TgGC, completely blocking parasite motility and infectivity (6, 8). Furthermore, we determined that TgPKG functions by controlling secretion of microneme proteins that are required for parasite motility, host cell invasion, and host cell egress (6). However, the mechanisms by which TgPKG controls microneme secretion and motility remain unclear because its substrates have not been identified or characterized. We hypothesize that TgPKG regulates T. gondii motility and virulence through phosphorylation of proteins that regulate microneme expression and secretion. Here, we will utilize modern genetic, transcriptomic, and proteomic approaches to test our hypothesis with three independent Specific Aims. Specific Aim 1: Identify genes differentially expressed following TgPKG knockdown. TgPKG controls T. gondii infectivity by regulating microneme secretion (6). We speculate that TgPKG directly modifies proteins required for microneme fusion with the plasma membrane. However, TgPKG may also regulate microneme secretion by modulating gene expression since PKGs regulate several transcription factors in other organisms (9). Previously, we generated a T. gondii line that expresses TgPKG fused to an auxin-inducible degron that allows for rapid and robust depletion of TgPKG with auxin treatment (6). Here we will identify genes that are differentially expressed following conditional depletion of TgPKG using RNA-Seq. Specific Aim 2: Identify interactors of TgPKG under basal and activated conditions. As with most kinases, we predict that TgPKG transiently interacts with its protein substrates following activation. We will use liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify TgPKG interactors captured by TurboID biotin-proximity labeling (10) and co-immunoprecipitation. We will treat extracellular parasites with or without a cell permeable cGMP analog (PET-cGMP) to distinguish proteins that interact with TgPKG basally or following activation. We have already generated an epitope-tagged TgPKG line for co-immunoprecipitation experiments (6) and a new TurboID-tagged TgPKG line for kinetic proximity labeling experiments. These two approaches are complementary since TurboID will provide a snapshot of proteins in close proximity (<15 nm) to TgPKG in live parasites, while co-immunoprecipitation will reveal proteins that directly interact with TgPKG. Furthermore, we will simultaneously assess the phosphorylation status each peptide identified, which could be modulated upon TgPKG activation if it is a direct substrate. Specific Aim 3: Define the TgPKG-dependent phosphoproteome under basal and activated conditions. In addition to knowing what TgPKG directly interacts with, it is critical to determine which interactors are phosphorylated by TgPKG. Using the TgPKG knockdown line, we will use stable isotope labeling using amino acids in cell culture (SILAC) phosphoproteomics to identify and quantify phosphopeptides from extracellular parasites treated with or without PET-cGMP in the presence or absence of TgPKG (+/- auxin treatment). Overall Impact. This study will provide critical insights into how TgPKG controls T. gondii motility and expose additional essential targets for drug development for treating toxoplasmosis. Since PKGs are conserved and essential in other apicomplexan parasites, like Plasmodium (11), this study will likely serve as a model for apicomplexan PKG function. Candidate substrates of TgPKG will be comprehensively investigated further as the subject of NIH R01 proposals going forward.

具体目标 由于没有疫苗可预防，全球25-30%的人患有弓形虫病 感染和目前的治疗是非治愈性的（1）。弓形虫病的一线治疗包括 乙胺嘧啶和磺胺嘧啶，它们抑制细胞内寄生虫复制，但毒性很高， 消除T.弓形虫（2）。因此，迫切需要新的无毒 阻断T.急性（速殖子）和慢性（缓殖子）生命阶段的弓形虫感染性。 环磷酸鸟苷（cGMP）已成为T。弓形虫感染性 协调寄生虫运动（3，4）。之前，我们确定T。刚地鸟苷酸环化酶 从GTP合成cGMP以刺激宿主细胞进出的运动性（5）。有条件耗竭 TgGC麻痹T.弓形虫，使寄生虫不能建立感染和引起疾病。 此外，我们还确定了质膜上的cGMP依赖性蛋白激酶（TgPKG）作为一种蛋白激酶， cGMP在T.速殖子（6）和缓殖子（7）均为弓形虫。化学抑制或 条件性敲除TgPKG表型模拟TgGC的丢失，完全阻断寄生虫运动性和感染性 （6，8）。此外，我们确定TgPKG通过控制微线体蛋白的分泌发挥功能， 寄生虫运动、宿主细胞侵入和宿主细胞排出所需的蛋白质（6）。然而， TgPKG控制微线体分泌和运动仍然不清楚，因为它的底物还没有被发现。 识别或表征。我们假设TgPKG调节T.弓形虫运动和毒力通过 调节微线表达和分泌的蛋白质的磷酸化。在这里，我们将利用现代 遗传学、转录组学和蛋白质组学方法，以三个独立的特定目的来检验我们的假设。 具体目标1：鉴定TgPKG敲低后差异表达的基因。TgPKG对照T. 通过调节微线体分泌来提高弓形虫感染性（6）。我们推测TgPKG直接修饰蛋白质 微线体与质膜融合所必需的。然而，TgPKG也可能调节微线体 由于PKG调节其他生物体中的几种转录因子，因此通过调节基因表达来分泌 （九）、之前，我们生成了一个T。表达TgPKG融合到生长素诱导降解决定子的弓形虫品系， 允许用生长素处理快速和稳健地消耗TgPKG（6）。在这里，我们将确定基因， 使用RNA-Seq在TgPKG条件性耗竭后差异表达。 具体目标2：在基础和活化条件下鉴定TgPKG的相互作用物。与大多数激酶一样， 我们预测TgPKG在激活后与其蛋白质底物瞬时相互作用。我们将使用液体 色谱-串联质谱（LC-MS/MS）识别TurboID捕获的TgPKG相互作用物 生物素邻近标记（10）和免疫共沉淀。我们将治疗细胞外寄生虫， 细胞渗透性cGMP类似物（PET-cGMP），以区分与TgPKG基础或后续相互作用的蛋白质 activation.我们已经建立了一个表位标记的TgPKG系用于免疫共沉淀实验 (6)和用于动力学邻近标记实验的新TurboID标记的TgPKG系。这两种方法 这是互补的，因为TurboID将提供与活体中的TgPKG非常接近（<15 nm）的蛋白质的快照。 寄生虫，而免疫共沉淀将揭示与TgPKG直接相互作用的蛋白质。而且我们 将同时评估每种鉴定的肽的磷酸化状态， TgPKG激活（如果是直接底物）。 具体目标3：定义基础和活化条件下的TgPKG依赖性磷酸化蛋白质组。 除了知道TgPKG直接与什么相互作用之外，确定哪些相互作用者是关键的。 通过TgPKG磷酸化。使用TgPKG敲低系，我们将使用稳定同位素标记，使用氨基标记。 细胞培养物中的磷酸（SILAC）磷酸蛋白质组学，以识别和定量细胞外 在存在或不存在TgPKG（+/-生长素处理）的情况下用或不用PET-cGMP处理的寄生虫。 整体影响。本研究将为TgPKG如何控制T。弓形虫活动和暴露 用于治疗弓形虫病的药物开发的额外的基本靶标。由于PKG是保守的， 在其他顶复门寄生虫中是必需的，如疟原虫（11），这项研究可能会作为一个模型， apicomplexan PKG功能。TgPKG的候选底物将进一步全面研究， NIH R 01提案的主题。