The Role of Clathrin in Post-Golgi Trafficking in Toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle.

顶复门寄生虫（Apicomplexan parasites）为一类单细胞真核生物，拥有高度极性化的分泌系统，其包含宿主细胞入侵所必需的两类特异性分泌细胞器：微线体（micronemes）与棒状体（rhoptries）。与之相对，这类寄生虫的内体系统（endosomal system）的功能目前仍知之甚少。因缺乏多种经典的内吞作用相关因子，我们推测其内吞过程仅依赖网格蛋白介导的转运机制。有趣的是，在刚地弓形虫（Toxoplasma gondii）中，我们仅能在高尔基体（Golgi）处检测到内源性网格蛋白重链1（clathrin heavy chain 1，CHC1），而无法在寄生虫的细胞表面观察到该蛋白的分布。为了对刚地弓形虫CHC1开展功能表征，我们构建了可条件表达显性负效网格蛋白Hub片段的寄生虫突变株，并通过实验证实CHC1对于反式高尔基体网络（trans-Golgi network，TGN）处的囊泡形成不可或缺。由此可见，CHC1的功能缺失会引发高尔基体形态异常，阻断特异性分泌细胞器微线体、棒状体以及表膜（pellicle）的生物发生进程。但我们未在该寄生虫中观察到网格蛋白介导内吞作用的形态学证据，因此推测这类寄生虫已重塑其囊泡转运系统，以适应专性胞内寄生的生活方式。