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，这是一种对发育中的胎儿有潜在生命威胁的疾病， 免疫力低下的人。目前治疗弓形虫病的药物常引起不良反应， 导致治疗中断的影响，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T.弓形虫，因为运动性对毒力至关重要。T. 弓形虫利用其独特的细胞运动形式侵入宿主细胞，穿过许多生物屏障， 在整个宿主体内传播。一种非传统的肌球蛋白运动蛋白TgMyoA驱动运动， 这个项目将测试运动功能和寄生虫运动受钙调节的假设， 介导的TgMyoA磷酸化。目的1旨在研究钙依赖性心肌细胞的运动性， 蛋白激酶（TgCDPK 3），其最近显示磷酸化TgMyoA。最近开发的3D运动 将使用测定来确定TgCDPK 3的破坏对运动性的特异性影响，并且重组 TgMyoA表达以确定磷酸化如何影响运动功能。由于TgCDPK 3磷酸化许多 除了TgMyoA之外，Aim 1还将确定是否用TgMyoA表达寄生虫蛋白。 在通常被TgCDPK 3磷酸化的位点上的磷酸化模拟氨基酸足以克服TgCDPK 3的磷酸化作用。 在缺乏功能性TgCDPK 3的寄生虫中观察到的运动缺陷。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则振荡中起作用 在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， （例如，与咖啡因，延长钙振荡），以确定是否速度 振荡是相关的和/或因果的。结合起来，这两个目标将提供新的见解， 钙和TgMyoA磷酸化在运动调节中的作用。了解TgMyoA的调节将是 这对于确定如何最有效地靶向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.