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: 10171739
• 负责人: Rachel Stadler
• 金额: $ 3.1万
• 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/10171739
• 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.

项目摘要 Toxoplasma Gondii是一种原生动物寄生虫，感染了世界人口的30-50％。寄生虫 引起弓形虫病，这是一种潜在的胎儿威胁生命的疾病， 免疫功能低下的个体。目前可用的药物治疗弓形虫病通常会导致不利的一面 导致停用治疗的影响，突出了对新治疗的需求。一 药物开发的潜在靶标是T. gondii的母系，因为运动对于病毒至关重要。 T. Gondii使用其独特的细胞运动形式来侵入宿主细胞，穿越许多生物屏障，然后 在整个宿主生物体中传播。一种非常规的肌球蛋白运动蛋白TGMYOA驱动运动性， 该项目将检验以下假设，即运动功能和寄生虫运动受到钙调节 TGMYOA介导的磷酸化。 AIM 1试图研究钙依赖性运动中的作用 蛋白激酶（TGCDPK3）最近显示出磷酸化的TGMYOA。最近开发的3D运动 测定将用于确定TGCDPK3破坏对运动的特定效果和重组 TGMYOA表达以确定磷酸化如何影响运动功能。随着TGCDPK3磷酸化许多 除TGMYOA外 通常由TGCDPK3磷酸化的位点的磷酸化氨基酸足以克服 在缺乏功能性TGCDPK3的寄生虫中看到的运动缺陷。 AIM 2将确定寄生虫中的振荡是否 在运动过程中观察到的细胞内钙水平在降低寄生虫的常规振荡中起作用 速度在3D中可见。使用表达钙指标的寄生虫，钙振荡将被改变 药理学（例如，使用咖啡因，可以延长钙振荡）以确定速度是否是否 振荡是相关性和/或结构化的。结合在一起，这两个目标将为角色的作用提供新颖的见解 运动调节中的钙和TGMYOA磷酸化。了解TGMYOA的调节将是 对于确定如何最有效地靶向药物开发的运动至关重要。