A Small-Molecule Inhibitor of <em>T. gondii</em> Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.刚地弓形虫（Toxoplasma gondii）是一种专性细胞内寄生虫，可通过主动侵入过程进入宿主细胞。宿主细胞侵入与寄生虫运动由一套已知包含四种蛋白的肌球蛋白马达复合物（myosin motor complex）驱动：非常规XIV类肌球蛋白TgMyoA、肌球蛋白轻链TgMLC1，以及两种膜相关蛋白TgGAP45与TgGAP50。目前学界对该肌球蛋白马达复合物的活性调控机制所知甚少。本研究发现，使用近期鉴定出的一种入侵与运动小分子抑制剂处理寄生虫后，TgMLC1的电泳迁移率会发生快速且不可逆的改变。尽管尚未明确TgMLC1修饰的具体分子本质，但该修饰位点已被定位在Val46-Arg59肽段。为了探明TgMLC1的修饰是否是化合物处理后寄生虫出现运动缺陷的原因，研究人员通过体外运动实验（in vitro motility assay）对比了对照组与化合物处理组寄生虫的肌球蛋白马达复合物活性。相较于对照组复合物，携带修饰后TgMLC1的TgMyoA马达复合物的运动活性显著降低。这一活性变化很可能解释了化合物处理后寄生虫的运动缺陷，同时也是首个在任何物种中证实XIV类肌球蛋白的机械活性可通过其结合轻链的翻译后修饰（posttranslational modifications）进行调控的研究证据。