Regulation of host cell egress by Toxoplasma gondii

弓形虫对宿主细胞出口的调节

• 批准号: 10640220
• 负责人: L. David Sibley
• 金额: $ 61.44万
• 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/10640220
• 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; Invaded; 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; calcium-dependent protein kinase; candidate identification; cell motility; chronic infection; design; extracellular; human disease; in vitro activity; in vivo; inhibitor; knock-down; mutant; novel therapeutic intervention; permissiveness; 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.

项目摘要/摘要 Apicomplexan寄生虫的主动运动控制宿主细胞的侵袭和出口，这对于对 感染的细胞内周期。这些过程受钙调节的粘合剂分泌控制 来自微元素的蛋白质，以及位于下面 寄生虫质膜。先前在弓形虫的研究确定了钙的重要性 依赖性蛋白激酶和蛋白激酶G（PKG）在控制微原质分泌，运动，出口， 和入侵。该级联反应受到严格调节，最近的研究还表明 蛋白激酶A（PKAC1）会抑制钙信号传导以关闭MicroNeme分泌并损害运动 入侵完成后。 PKG与PKAC1活动之间的平衡控制着激活的决定 运动性并促进出口与保持细胞内。虽然PKAC1在阻止过早的作用中的作用 出口很明显，它的调节机理也不是它磷酸化以抑制的目标 钙信号传导和阻塞出口已被阐明。 PKA活动受环状产生的控制 腺苷酸环化酶（ACS）的AMP（CAMP）及其消耗磷酸二酯酶（PDES）。在初步 研究，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，以定义它们在时间和空间调节PKA活动的方式。我们也有 使用蛋白质降解方法来创建有条件敲低的PKA同工型并验证PKAC1 控制II型寄生虫中的出口。我们将使用严格调节的条件敲低方法 研究PKAC1的下游靶标，该靶标的结合 磷蛋白质组学研究以鉴定底物和下游验证研究，以测试其在 调节出口。我们将验证特定磷酸化位点在PKAC1靶标中的作用 钙信号，运动和出口。总体而言，拟议的研究将阐明分子途径 PKAC1下降调节钙水平，微米分泌和动力以防止出口，从而 平衡PKG的激活效应。阐明PKG/PKA激酶的分子调节 T. gondii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 用于治疗学的发展。

项目成果

The Toxoplasma micropore mediates endocytosis for selective nutrient salvage from host cell compartments.

• DOI: 10.1038/s41467-023-36571-4
• 发表时间: 2023-02-22
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Wan, Wenyan;Dong, Hui;Lai, De-Hua;Yang, Jiong;He, Kai;Tang, Xiaoyan;Liu, Qun;Hide, Geoff;Zhu, Xing-Quan;Sibley, L. David;Lun, Zhao-Rong;Long, Shaojun
• 通讯作者: Long, Shaojun