Small Molecule Approaches to Studying T. gondii Invasion

描述(申请人提供)：弓形虫是一种分布广泛的尖端复合体寄生虫，可在先天性感染的胎儿和免疫功能低下的人中引起严重疾病。弓形虫在感染过程中使用一种独特的滑动运动来入侵宿主细胞并在体内传播，运动对寄生虫的毒力是必不可少的。为滑动运动提供动力的机制在顶复合体寄生虫中保存得很好，但人们对运动机制核心的非传统肌球蛋白马达蛋白是如何调控的知之甚少。最近的高通量筛选确定了24种新的小分子抑制物和6种弓形虫侵袭宿主细胞的增强剂。引人注目的是，在24个抑制入侵的小分子中，有21个被发现抑制了寄生虫的运动，所有6个入侵促进剂都增强了寄生虫的运动。这项提案的重点是最有希望的运动抑制剂之一，化合物115556的详细作用机制。当化合物115556被添加到弓形虫中时，它导致与驱动运动的肌球蛋白马达(TgMyoA)相关的单个肌球蛋白轻链(TgMLc1)经历了显著的凝胶迁移率变化，这是由于一种尚不具特征的翻译后修饰。体外运动试验显示，含有修饰的TgMLC1的弓形虫运动复合体表现出运动活性降低。在这项工作中，TgMLC1也被证明是磷酸化和二甲基化的。该项目的中心假设是，TgMLc1的翻译后修饰，包括115556诱导的修饰，调节TgMyoA马达的功能和寄生虫的运动。该项目的具体目标是：(1)确定115556诱导的TgMLc1修饰的性质及其对TgMyoA功能的影响，并直接验证115556诱导的TgMLc1修饰负责化合物抑制寄生虫运动的假设；(2)确定115556的靶点。初步数据表明，115556不与TgMLc1结合并直接作用于TgMLc1。将确定115556的直接靶标，并阐明将该靶标与TgMLc1修饰联系起来的途径；(3)确定TgMLc1磷酸化和甲基化对TgMyoA活性和寄生虫运动性的功能后果，并确定高通量筛选中是否有任何其他运动抑制物或增强剂通过TgMLc1的磷酸化或甲基化起作用。这项工作将为了解弓形虫移动和入侵宿主细胞所涉及的蛋白质和途径提供新的见解。更好地了解弓形虫和其他顶复合体寄生虫的运动性是如何调节的，可能会导致新的化疗方法来治疗这些寄生虫引起的毁灭性疾病。

DESCRIPTION (provided by applicant): Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in the congenitally-infected fetus and in immunocompromised persons. T. gondii uses a unique form of gliding motility to invade host cells and disseminate through the body during infection, and motility is essential to parasite virulence. The machinery that powers gliding motility is well conserved in apicomplexan parasites, but very little is known about how the unconventional myosin motor protein at the heart of the motility machinery is regulated. A recent high-throughput screen identified 24 novel small-molecule inhibitors and six enhancers of host cell invasion by T. gondii. Strikingly, 21 of the 24 small molecules that inhibit invasion were found to inhibit parasite motility, and all six of the invasion enhancers enhance parasite motility. This proposal is focused on the detailed mechanism of action of one of the most promising of the motility inhibitors, compound 115556. When compound 115556 is added to T. gondii, it causes the single myosin light chain (TgMLC1) associated with the myosin motor that drives motility (TgMyoA) to undergo a pronounced electrophoretic mobility shift, due to an as yet uncharacterized posttranslational modification. Isolated T. gondii motor complexes containing the modified TgMLC1 show decreased motor activity in an in vitro motility assay. In the course of this work, TgMLC1 was also shown to be phosphorylated and dimethylated. The central hypothesis of this project is that posttranslational modifications of TgMLC1, including the modification induced by 115556, regulate the function of the TgMyoA motor and parasite motility. The Specific Aims of the project are to: (1) Determine the nature of the 115556-induced modification to TgMLC1 and its effect on TgMyoA function and directly test the hypothesis that the 115556-induced modification of TgMLC1 is responsible for the inhibition of parasite motility by the compound; (2) Identify the target of 115556. The preliminary data suggest that 115556 does not bind to and act directly on TgMLC1. The direct target of 115556 will be identified, and the pathway connecting this target to the modification of TgMLC1 elucidated; (3) Determine the functional consequences of TgMLC1 phosphorylation and methylation on TgMyoA activity and parasite motility, and establish whether any of the other motility inhibitors or enhancers identified in the high-throughput screen act through phosphorylation or methylation of TgMLC1. This work will provide new insights into the proteins and pathways involved in the motility and invasion of host cells by T. gondii. A greater understanding of how motility is regulated in T. gondii and other apicomplexan parasites is likely to lead to new chemotherapeutic approaches to treating the devastating diseases these parasites cause.