Small Molecule Approaches to Studying T. gondii Invasion

描述（由申请人提供）：弓形虫是一种广泛分布的顶复门寄生虫，可导致先天性感染胎儿和免疫功能低下者发生严重疾病。T.在感染过程中，弓形虫利用一种独特的滑行运动形式侵入宿主细胞并在体内传播，运动对寄生虫的毒力至关重要。在顶复门寄生虫中，为滑行运动提供动力的机制是保守的，但对运动机制核心的非常规肌球蛋白运动蛋白是如何调节的知之甚少。最近的高通量筛选鉴定了24种新的小分子抑制剂和6种T.刚地。引人注目的是，24种抑制入侵的小分子中有21种被发现抑制寄生虫的运动，而所有六种入侵增强剂都增强寄生虫的运动。该建议的重点是详细的行动机制的最有前途的运动抑制剂之一，化合物115556。 当化合物115556加入T.在弓形虫中，由于一种尚未表征的翻译后修饰，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（TgMLC 1）发生明显的电泳迁移率变化。孤立T.含有修饰的TgMLC 1的弓形虫运动复合体在体外运动性测定中显示降低的运动活性。在这项工作的过程中，TgMLC 1也被证明是磷酸化和二甲基化的。该项目的中心假设是TgMLC 1的翻译后修饰，包括由115556诱导的修饰，调节TgMyoA运动和寄生虫运动的功能。 该项目的具体目的是：（1）确定115556诱导的TgMLC 1修饰的性质及其对TgMyoA功能的影响，并直接检验115556诱导的TgMLC 1修饰是化合物抑制寄生虫运动的原因的假设;（2）鉴别115556的靶点。初步数据表明，115556不结合并直接作用于TgMLC 1。将鉴定115556的直接靶点，并阐明将该靶点与TgMLC 1修饰连接的途径;（3）确定TgMLC 1磷酸化和甲基化对TgMyoA活性和寄生虫运动性的功能后果，并确定在高通量筛选中鉴定的任何其他运动性抑制剂或增强剂是否通过TgMLC 1的磷酸化或甲基化起作用。 这项工作将提供新的见解的蛋白质和途径参与的运动和入侵宿主细胞的T。刚地。更深入地了解T.弓形虫和其他顶复门寄生虫的研究可能会导致新的化学治疗方法，以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.