Is Calcium the Gear Shift? The Role of Calcium-Mediated TgMyoA Phosphorylation in Toxoplasma Gondii Motility

钙是变速杆吗？

• 批准号: 9911622
• 负责人: Rachel Stadler
• 金额: $ 3.05万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911622
• 关键词: 3-Dimensional; Acceleration; Affect; American; Amino Acids; Biological; Biological Assay; Biophysics; Caffeine; Calcium; Calcium Oscillations; Cause of Death; Cells; Clinical; Communicable Diseases; Complex; Data; Defect; Development; Disease; Drug Targeting; Etiology; Extracellular Matrix; Fetal Development; Fetus; Goals; Immunocompromised Host; Individual; Infant; Invaded; Lead; Life; Life Cycle Stages; Light; Measures; Mechanics; Mediating; Molecular Genetics; Motor; Movement; Mutate; Myosin ATPase; Organism; Parasites; Pattern; Periodicity; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Phosphorylation Site; Play; Population; Process; Proteins; Publishing; Recombinants; Regulation; Role; Site; Source; Structure; System; Techniques; Testing; Time; Toxoplasma gondii; Toxoplasmosis; United States; Video Microscopy; Virulence; acute infection; base; biophysical analysis; calcium indicator; calcium-dependent protein kinase; cell motility; drug development; foodborne; foodborne illness; improved; in vivo; insight; mathematical model; mutant; new therapeutic target; novel; novel strategies; side effect; software development; three-dimensional modeling; tool

Project Summary Toxoplasma gondii is a protozoan parasite that infects between 30-50% of the world’s population. The parasite causes toxoplasmosis, which is a potentially life-threatening disease in the developing fetus and immunocompromised individuals. Currently available drugs to treat toxoplasmosis often cause adverse side effects that lead to discontinuation of treatment, highlighting the need for new and improved treatments. One potential target for drug development is the motile system of T. gondii, as motility is essential for virulence. T. gondii uses its unique form of cellular motility to invade host cells, traverse numerous biological barriers, and disseminate throughout the host organism. An unconventional myosin motor protein, TgMyoA, drives motility, and this project will test the hypothesis that motor function and parasite motility are regulated by calcium- mediated phosphorylation of TgMyoA. Aim 1 seeks to investigate the role in motility of a calcium-dependent protein kinase (TgCDPK3) that was recently shown to phosphorylate TgMyoA. A recently developed 3D motility assay will be used to determine the specific effect(s) that disruption of TgCDPK3 has on motility, and recombinant TgMyoA expression to establish how phosphorylation affects motor function. As TgCDPK3 phosphorylates many parasite proteins in addition to TgMyoA, Aim 1 will also determine whether expressing TgMyoA with phosphomimetic amino acids at the sites normally phosphorylated by TgCDPK3 is sufficient to overcome the motility defects seen in parasites lacking functional TgCDPK3. Aim 2 will determine if the oscillations in parasite intracellular calcium levels observed during motility play a role in regulating the regular oscillations in parasite velocity seen in 3D. Using calcium indicator-expressing parasites, the calcium oscillations will be altered pharmacologically (e.g., with caffeine, which lengthens the calcium oscillations) to determine whether the velocity oscillations are correlation and/or causative. Combined, the two Aims will provide novel insights into the role of calcium and TgMyoA phosphorylation in motility regulation. Understanding the regulation of TgMyoA will be critical to determining how to most effectively target motility for drug development.

项目概要 弓形虫是一种原生动物寄生虫，感染世界上 30-50% 的人口。寄生虫 引起弓形体病，这是一种可能危及发育中胎儿生命的疾病 免疫功能低下的个体。目前治疗弓形体病的药物常常会引起不良副作用 导致治疗停止的影响，强调需要新的和改进的治疗方法。一 药物开发的潜在目标是弓形虫的运动系统，因为运动对于毒力至关重要。 T。 弓形虫利用其独特的细胞运动形式侵入宿主细胞，穿越众多的生物屏障，并 传播到整个宿主生物体。一种非常规的肌球蛋白运动蛋白 TgMyoA 可驱动运动， 该项目将检验运动功能和寄生虫运动受钙调节的假设 介导的 TgMyoA 磷酸化。目标 1 旨在研究钙依赖性细胞在运动中的作用 最近被证明可以磷酸化 TgMyoA 的蛋白激酶 (TgCDPK3)。最近开发的 3D 运动 测定将用于确定 TgCDPK3 破坏对运动和重组的具体影响 TgMyoA 表达以确定磷酸化如何影响运动功能。由于 TgCDPK3 磷酸化许多 除了 TgMyoA 之外，Aim 1 还将确定是否表达 TgMyoA 的寄生虫蛋白 通常被 TgCDPK3 磷酸化的位点上的拟磷酸氨基酸足以克服 缺乏功能性 TgCDPK3 的寄生虫存在运动缺陷。目标 2 将确定寄生虫中的振荡是否 运动过程中观察到的细胞内钙水平在调节寄生虫的规律振荡中发挥作用 3D 中看到的速度。使用表达钙指示剂的寄生虫，钙振荡将被改变 从药理学上（例如，咖啡因，它会延长钙振荡）来确定速度是否 振荡是相关的和/或因果的。结合起来，这两个目标将为以下角色提供新的见解： 钙和 TgMyoA 磷酸化在运动调节中的作用。了解 TgMyoA 的调节将有助于 对于确定如何最有效地靶向药物开发的动力至关重要。