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.

项目总结/摘要 顶复门寄生虫的主动运动控制宿主细胞的侵入和外出，这些步骤对宿主细胞的生长至关重要。 感染的细胞内循环。这些过程是由钙调节的粘附剂分泌控制的。 蛋白质从微线，连同协调行动的肌动蛋白肌球蛋白马达，位于下面的 寄生物质膜先前对刚地弓形虫的研究已经确定了钙的重要性， 依赖性蛋白激酶和蛋白激酶G（PKG）在控制微线体分泌、运动、外出 和入侵。这种级联反应受到严格调控，最近的研究也表明， 蛋白激酶A（PKAc 1）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PKAc 1活性之间的平衡决定了激活 运动性和促进排出相对于保持在细胞内。虽然PKAc 1在阻断早产儿的作用， 出口是明确的，无论是它的调节机制，也不是它磷酸化的目标，以抑制 已经阐明了钙信号传导和阻断出口。PKA的活性受环 腺苷酸环化酶（AC）对cAMP（cAMP）的作用及其被磷酸二酯酶（PDE）的消耗。初步 在研究中，我们已经鉴定了控制cAMP水平以调节PKA的候选AC。在拟议的研究中， 我们将探讨它们的功能，以确定它们如何在时间和空间上调节PKA活性。我们还 使用蛋白质降解方法来产生PKA同种型的条件性敲低，并验证PKAc 1 控制II型寄生虫的逸出。我们将使用严格监管的有条件击倒方法， 研究PKAc 1的下游靶点，这些靶点使用以下组合介导出口阻塞： 磷酸化蛋白质组学研究，以确定底物和下游验证研究，以测试其在 控制出口我们将验证PKAc 1靶点中特异性磷酸化位点在抑制细胞凋亡中的作用。 钙信号传导、运动和排泄。总的来说，拟议的研究将阐明 PKAc 1通过其下调钙水平、微线体分泌和运动性以防止外出，因此 平衡PKG的激活效果。阐明PKG/PKA激酶的分子调控， T.弓形虫将促进未来的研究，设计阻断这些途径的抑制剂，从而提供新的途径 用于开发治疗方法。