Regulation of host cell egress by Toxoplasma gondii

项目摘要/摘要 顶端复合体寄生虫的主动运动控制宿主细胞的入侵和输出，这些步骤对 感染的细胞内循环。这些过程是由钙调节的粘合剂的分泌所控制的。 来自微线的蛋白质，以及位于肌球蛋白下方的肌动蛋白-肌球蛋白马达的协同作用 寄生虫质膜。以前对弓形虫的研究已经确定了钙的重要性- 依赖的蛋白激酶和蛋白激酶G(PKG)在控制微线粒的分泌、运动、排出、 和入侵。这一级联反应受到严格调控，最近的研究也表明， 蛋白激酶A(PKAc1)抑制钙信号，阻断微线体分泌，损害运动能力 在入侵完成后。PKG和PKAc1活动之间的平衡决定了激活的决定 运动和促进出口，而不是保持细胞内。尽管PKAc1在阻断早产中的作用 出口是明确的，既不是它的调节机制，也不是它为了抑制而磷酸化的目标 钙信号和阻断出口已被阐明。PKA活性受环状蛋白的产生控制 腺苷环化酶(ACS)的AMP(CAMP)和磷酸二酯酶(PDE)对其的消耗。在预赛中 研究表明，我们已经确定了控制cAMP水平以调节PKA的候选ACS。在拟议的研究中， 我们将探索它们的功能，以确定它们如何在时间和空间上调节PKA活动。我们还有 使用蛋白质降解方法创建有条件的PKA亚型敲除，并验证PKAc1 控制II型寄生虫的出口。我们将使用严格监管的有条件淘汰方法来 使用以下组合调查调节出口阻断的PKAc1的下游目标 鉴定底物的磷酸蛋白质组学研究和测试它们在其中的作用的下游验证研究 监管出口。我们将验证PKAc1靶标中特定的磷酸化位点在抑制 钙信号、运动性和出口。总的来说，拟议的研究将阐明分子途径。 PKAc1通过下调钙水平、微线粒分泌和运动性来防止外流，从而 抵消PKG的激活作用。PKG/PKA蛋白激酶分子调控机制的研究 弓形虫将促进未来的研究，以设计阻断这些途径的抑制剂，从而提供新的途径 用于治疗学的发展。

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.