Regulation of host cell egress by Toxoplasma gondii

弓形虫对宿主细胞出口的调节

• 批准号: 10441782
• 负责人: L. David Sibley
• 金额: $ 62.16万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-07 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10441782
• 关键词: Actins; Adenylate Cyclase; Adhesives; Auxins; Binding; Biotin; CRISPR/Cas technology; Calcium; Calcium Signaling; Cell membrane; Cells; Code; Consumption; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Data; Development; Dominant-Negative Mutation; Down-Regulation; Equilibrium; Fostering; Foundations; Future; Genetic Transcription; Goals; Immune system; Immunocompromised Host; Impairment; In Vitro; Individual; Infection; Knock-out; Knowledge; Label; Ligation; Lytic Phase; Mass Spectrum Analysis; Mediating; Membrane; Molecular; Motor; Myosin ATPase; Parasite Control; Parasites; Pathway interactions; Persons; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Process; Production; Protein Isoforms; Proteins; Regulation; Risk; Role; Structure; Testing; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; active control; base; calcium-dependent protein kinase; cell motility; chronic infection; design; extracellular; human disease; in vitro activity; in vivo; inhibitor; knock-down; mutant; novel therapeutic intervention; phosphoproteomics; phosphoric diester hydrolase; premature; prevent; protein degradation; protein kinase A kinase; therapeutic development; validation studies

Project Summary/Abstract Active motility by apicomplexan parasites controls host cell invasion and egress, steps that are critical to the intracellular cycle of infection. These processes are governed by calcium-regulated secretion of adhesive proteins from micronemes, together with the concerted action of an actin-myosin motor that lies beneath the parasite plasma membrane. Prior studies in Toxoplasma gondii have established the importance of calcium- dependent protein kinases and protein kinase G (PKG) in controlling microneme secretion, motility, egress, and invasion. This cascade is tightly regulated and recent studies have also shown that the c1 isoform of protein kinase A (PKAc1) dampens calcium signaling to shut down microneme secretion and impair motility after invasion is complete. The balance between PKG vs. PKAc1 activities governs the decision to activate motility and promote egress vs. to remain intracellular. Although the role of PKAc1 in blocking premature egress is clear, neither its mechanism of regulation nor the targets that it phosphorylates in order to dampen calcium signaling and block egress have been elucidated. PKA activity is governed by production of cyclic AMP (cAMP) by adenylate cyclases (ACs) and its consumption by phosphodiesterases (PDEs). In preliminary studies, we have identified candidate ACs that control cAMP levels to regulate PKA. In the proposed studies, we will explore their functions to define how they temporally and spatially regulate PKA activity. We have also used a protein degradation approach to create conditional knockdowns of PKA isoforms and verify that PKAc1 controls egress in type II parasites. We will use the tightly regulated conditional knockdown approach to investigate downstream targets of PKAc1 that mediate the egress block using a combination of phosphoproteomic studies to identify substrates and downstream validation studies to test their roles in regulating egress. We will validate the role of specific phosphorylation sites in PKAc1 targets in suppressing calcium signaling, motility, and egress. Collectively, the proposed studies will elucidate the molecular pathway by which PKAc1 down regulates calcium levels, microneme secretion, and motility to prevent egress, thus counterbalancing the activating effects of PKG. Elucidating the molecular regulation of the PKG/PKA kinases in T. gondii will foster future studies to design inhibitors that block these pathways, thus providing new avenues for development of therapeutics.

项目概要/摘要 顶复门寄生虫的主动运动控制宿主细胞的侵入和流出，这些步骤对于宿主细胞的入侵和流出至关重要。 细胞内感染周期。这些过程受到钙调节的粘合剂分泌的控制 来自微线体的蛋白质，以及位于微线体下方的肌动蛋白-肌球蛋白马达的协同作用 寄生虫质膜。先前对弓形虫的研究已经确定了钙的重要性 依赖蛋白激酶和蛋白激酶 G (PKG) 控制微线体的分泌、运动、排出、 和入侵。该级联受到严格调控，最近的研究还表明，c1 亚型 蛋白激酶 A (PKAc1) 抑制钙信号传导，从而关闭微线体分泌并损害运动性 入侵完成后。 PKG 与 PKAc1 活性之间的平衡决定激活的决定 运动并促进排出与保留在细胞内。尽管 PKAc1 在阻断早产儿中的作用 出口是明确的，无论是它的调节机制还是它磷酸化以抑制的目标 钙信号传导和阻断出口已被阐明。 PKA 活性受循环生成的控制 腺苷酸环化酶 (AC) 产生的 AMP (cAMP) 及其被磷酸二酯酶 (PDE) 消耗。在初步 研究中，我们已经确定了控制 cAMP 水平以调节 PKA 的候选 AC。在拟议的研究中， 我们将探索它们的功能，以确定它们如何在时间和空间上调节 PKA 活性。我们还有 使用蛋白质降解方法来创建 PKA 同工型的条件敲低并验证 PKAc1 控制 II 型寄生虫的出口。我们将使用严格监管的条件击倒方法 使用以下组合研究介导出口阻断的 PKAc1 下游目标 用于鉴定底物的磷酸蛋白质组学研究和用于测试其作用的下游验证研究 调节出口。我们将验证 PKAc1 靶标中特定磷酸化位点在抑制 钙信号传导、运动和排出。总的来说，拟议的研究将阐明分子途径 PKAc1 通过下调钙水平、微线体分泌和运动来防止流出，从而 抵消 PKG 的激活作用。阐明 PKG/PKA 激酶的分子调控 弓形虫将促进未来的研究，以设计阻断这些途径的抑制剂，从而提供新的途径 用于治疗方法的开发。