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

项目摘要 弓形虫是一种原生动物寄生虫，感染了世界上30%-50%的人口。寄生虫 引起弓形虫病，这是一种在发育中的胎儿中潜在威胁生命的疾病， 免疫功能受损的个体。目前治疗弓形虫病的药物往往会引起不良反应。 导致停止治疗的影响，突出了需要新的和改进的治疗方法。一 药物开发的潜在靶点是弓形虫的运动系统，因为运动对毒力是必不可少的。T. 弓形虫利用其独特的细胞运动形式入侵宿主细胞，穿越无数生物屏障，并 在整个寄主生物体中传播。一种非传统的肌球蛋白马达蛋白，TgMyoA，驱动运动， 这个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的磷酸化。目的1研究钙依赖蛋白在运动中的作用。 蛋白激酶(TgCDPK3)是最近发现的一种磷酸化TgMyoA的蛋白。一种最近开发的3D运动学 用比色法测定TgCDPK3的干扰对运动的特异性影响(S)，并重组 TgMyoA的表达以确定磷酸化如何影响运动功能。随着TgCDPK3磷酸化许多 除了TgMyoA外，寄生虫蛋白Aim 1还将决定TgMyoA与 TgCDPK3正常磷酸化位点上的仿磷氨基酸足以克服TgCDPK3 缺乏TgCDPK3功能的寄生虫的运动性缺陷。目标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调节寄生虫的规律性振荡中起着作用 在3D中看到的速度。利用表达钙指示剂的寄生虫，钙的振荡将被改变 药理学上(例如，咖啡因，它延长了钙振荡)以确定是否速度 振荡是相关的和/或起因的。这两个目标结合在一起，将为我们提供对 钙和TgMyoA在运动调节中的磷酸化。了解TgMyoA的调节将是 对于确定如何最有效地针对药物开发的运动性至关重要。

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.