Elucidation of TgPKG kinase substrates required for Toxoplasma motility

具体目标 弓形虫在全世界25%-30%的人类中持续存在，因为没有疫苗可以预防 感染和目前的治疗方法是无法治愈的(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的复制，但毒性很高，无法 消除弓形虫休眠组织包囊阶段(2)。因此，迫切需要新的无毒 在急性(速殖子)和慢性(慢殖子)生命阶段阻断弓形虫传染性的药物。 环鸟苷一磷酸(CGMP)已成为弓形虫感染性的主要调节因子。 协调寄生虫活动(3，4)。此前，我们确定了弓形虫鸟苷环化酶(TgGC) 从GTP合成cGMP以刺激宿主细胞的进出运动(5)。有条件地耗尽 TgGC使弓形虫瘫痪，使寄生虫无法建立感染和致病能力。 此外，我们确定质膜上的cGMP依赖的蛋白激酶(TgPKG)起着 CGMP在弓形虫速殖子(6例)和缓殖子(7例)中的中枢效应。化学抑制或 条件性敲除TgPKG表型缺失TgGC，完全阻断寄生虫的运动和感染性 (6、8)。此外，我们确定TgPKG通过控制微线蛋白的分泌发挥作用 寄生虫活动、宿主细胞入侵和宿主细胞出口所需(6)。然而，通过这些机制 TgPKG控制微线粒的分泌和运动仍不清楚，因为它的底物还没有 确定的或具有特征的。我们推测TgPKG通过调节弓形虫的运动和毒力 调节微线体表达和分泌的蛋白质的磷酸化。在这里，我们将利用现代 用遗传学、转录学和蛋白质组学的方法来验证我们的假设，有三个独立的特定目标。 具体目标1：确定TgPKG基因敲除后差异表达的基因。TgPKG控制T. 调节微线体分泌的弓形虫感染性(6)。我们推测TgPKG直接修饰蛋白质 微丝与质膜融合所需的。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 (9)。在此之前，我们产生了一个弓形虫细胞系，它表达TgPKG与生长素诱导的可降解蛋白 允许生长素治疗使TgPKG迅速而有力地耗尽(6)。在这里，我们将识别出 使用RNA-Seq条件耗尽TgPKG后的差异表达。 具体目标2：确定基础和激活条件下TgPKG的相互作用因子。与大多数激动酶一样， 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 LC-MS/MS鉴定TurboID捕获的TgPKG相互作用子 生物素-接近标记(10)和免疫共沉淀。我们将治疗胞外寄生虫，无论有没有 细胞通透性cGMP类似物(PET-cGMP)用于区分与TgPKG碱基或以下相互作用的蛋白质 激活。我们已经产生了一个表位标记的TgPKG品系，用于免疫共沉淀实验 (6)和一种新的TurboID标记的TgPKG系，用于动力学邻近标记实验。这两种方法是 互补，因为TurboID将提供与TgPKG非常接近(15 Nm)的活体蛋白质的快照 而免疫共沉淀将揭示与TgPKG直接相互作用的蛋白质。此外，我们 将同时评估每个识别的多肽的磷酸化状态，这可能是调节的 如果是直接底物，则激活TgPKG。 具体目标3：确定基础条件和激活条件下依赖TgPKG的磷酸蛋白质组。 除了知道TgPKG直接与什么交互外，确定哪些交互因素是至关重要的 被TgPKG磷酸化。使用TgPKG击倒线，我们将使用氨基稳定同位素标记 细胞培养中的酸(SILAC)磷酸蛋白质组学用于鉴定和定量细胞外的磷酸肽 在有或没有TgPKG(+/-生长素治疗)的情况下，用或不用PET-cGMP治疗寄生虫。 整体影响。这项研究将为TgPKG如何控制弓形虫的运动和暴露提供关键的见解 治疗弓形虫病药物开发的其他基本目标。由于PKG是保守的，并且 这项研究在其他顶端复合体寄生虫中是必不可少的，如疟原虫(11)，这项研究可能会成为 顶端复合体PKG功能。将进一步全面研究TgPKG的候选底物，因为 未来NIH R01提案的主题。

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.