A <em>Toxoplasma</em> Palmitoyl Acyl Transferase and the Palmitoylated Armadillo Repeat Protein TgARO Govern Apical Rhoptry Tethering and Reveal a Critical Role for the Rhoptries in Host Cell Invasion but Not Egress

Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.顶复门寄生虫（Apicomplexans）是一类专性细胞内寄生虫，可主动侵入宿主细胞以构建适于增殖的细胞内微环境。学界普遍认为，入侵的定向过程由棒状体（rhoptries）介导——这类特化的顶端分泌细胞器可将蛋白复合物注入宿主细胞，形成供寄生虫入侵的紧密连接。目前，对于调控棒状体生物发生、寄生虫顶端亚细胞组织以及入侵过程中该细胞器后续释放的分子因素，人们仍知之甚少。我们已鉴定出刚地弓形虫（Toxoplasma）中的棕榈酰酰基转移酶（palmitoyl acyltransferase）TgDHHC7，该酶定位于棒状体。值得注意的是，对TgDHHC7进行条件性敲低后，棒状体出现分散现象：它们无法在寄生虫顶端聚集，反而散布于整个细胞内。尽管这些棒状体的形态与组分看似正常，但无法在顶端锚定的结果是宿主细胞入侵被完全阻断。与之相反，敲低组的附着与逸出过程未受影响，这表明棒状体并非这些过程所必需。研究表明，TgDHHC7的棒状体靶向定位需要一段短小且高度保守的C端区域，而较大的变异N端结构域对于靶向定位与功能均非必需。此外，缺失被预测为酶活性关键残基的点突变体无法挽救顶端棒状体锚定缺陷，这强烈提示该细胞器的锚定依赖于TgDHHC7的棕榈酰化活性。我们还证实，棕榈酰化的仅含ARM重复序列（Armadillo Repeats-Only）棒状体蛋白（TgARO）是该活性的作用靶点：对TgARO进行条件性敲低可重现TgDHHC7敲低时出现的分散棒状体表型。顶复门寄生虫利用蛋白质棕榈酰化实现顶端细胞器锚定这一意外发现，为棒状体的生物发生与功能研究提供了新视角，也为针对刚地弓形虫及相关顶复门寄生虫的治疗干预开辟了新途径。