Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA.
PLoS Pathog. 2011 Oct;7(10):e1002280. doi: 10.1371/journal.ppat.1002280. Epub 2011 Oct 6.
Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.
顶复门寄生虫依赖一种新型的肌动蛋白依赖的运动形式，称为滑行运动，该运动依赖寄生虫肌动蛋白的聚合，以在宿主中迁移并侵入细胞。然而，寄生虫肌动蛋白在序列和功能上都有很大的差异，与传统肌动蛋白纤维的稳定性相比，只能形成短而不稳定的纤维。寄生虫肌动蛋白纤维不稳定性的分子基础及其与滑行运动的关系仍未解决。我们证明重组弓形虫（TgACTI）和疟原虫（PfACTI 和 PfACTII）肌动蛋白在体外聚合成长度非常短的纤维，但通过添加等摩尔水平的鬼笔环肽，可诱导其形成长而稳定的纤维。寄生虫肌动蛋白包含一个保守的鬼笔环肽结合位点，这是通过分子建模和计算对接确定的，然而，在几个残基上存在差异，这些残基预测会影响纤维的稳定性。特别是，鉴定出两个残基，它们在常规肌动蛋白纤维的不同亚基之间形成分子间接触，这些残基在顶复门寄生虫中表现出非保守的差异。用来自哺乳动物肌动蛋白的差异残基取代 TgACTI 中的差异残基，可导致在体外形成更长、更稳定的纤维。在刚地弓形虫中表达这些稳定的肌动蛋白，增加了对肌动蛋白稳定化合物 Jasplakinolide 的敏感性，并在没有治疗的情况下破坏了正常的滑行运动。这些结果确定了顶复门寄生虫中短而动态纤维的分子基础，并证明寄生虫肌动蛋白纤维固有的不稳定性是滑行运动的一个关键适应。
