Host cell manipulation by Toxoplasma

描述(申请人提供)：专性细胞内原生动物弓形虫是弓形虫脑炎的病原体，它仍然是艾滋病患者死亡的主要原因。目前可用的治疗弓形虫病的方法对弓形虫长期慢性感染没有疗效，导致活动性感染的复发。迫切需要新药。在哺乳动物细胞入侵期间，寄生虫产生一个特殊的寄生虫空泡(PV)，该空泡由含有寄生虫蛋白的独特膜与宿主细胞质隔开。弓形虫以广泛修改宿主细胞和将宿主细胞器重新路由到其PV而臭名昭著，主要是为了获取营养。寄生虫通常在寄主高尔基体周微管组织中心附近筑巢。这一区域位于内吞和生物合成途径的交汇处，而PV定位在细胞的这一区域可以促进囊泡运输的拦截，满足寄生虫对营养和脂膜的需求。为了支持这一假设，我们证明了弓形虫对来自溶酶体(例如胆固醇)和高尔基体(例如鞘脂)的宿主细胞脂有明显的需求。PV不与宿主细胞器融合，寄生虫从胞内和胞外细胞器取回脂质的机制仍然知之甚少。我们建议的总体目标是破译宿主溶酶体和弓形虫利用高尔基体的分子细节。我们以前的研究表明，弓形虫利用宿主微管网络将宿主溶酶体重定向到其液泡中，并将这些细胞器隔离在微管形成的PV膜内陷中。这一策略使寄生虫能够获得由内细胞级联细胞器提供的营养。然而，我们目前的模式仍然存在广泛的差距。具体目标1建议进行实验，以更好地了解弓形虫与宿主内吞途径的交集。我们将阐明弓形虫截获的内吞结构的性质和对寄生虫发育的贡献。我们将研究参与宿主微管-PV相互作用和PV膜转化的溶酶体隔离的分子机制。关于宿主高尔基体在弓形虫感染中的作用，人们几乎一无所知。我们的初步数据显示，寄生虫与高尔基体结合，将这个细胞器分裂成沿PV排列的高尔基体小片，并通过将分泌小泡吞噬到PV中来拦截高尔基体囊泡的运输。这一策略可能允许寄生虫从隔离的高尔基体小泡中取回高尔基体脂类物质。特定目的2结合实验验证了我们关于宿主-高尔基体-光伏相互作用的假设模型。我们将通过识别寄生虫靶标的宿主高尔基蛋白来研究导致宿主高尔基体破裂和高尔基体小泡传递到光伏的机制。我们将探索高尔基体重塑和寄生虫挽救脂肪之间的联系。我们的结果可能会提出基于对宿主细胞器功能的干扰来治疗弓形虫的新疗法的挑衅想法。

DESCRIPTION (provided by applicant): The obligate intracellular protozoan Toxoplasma gondii is the etiological agent of toxoplasmic encephalitis that remains a major cause of death in AIDS patients. The currently available treatment for toxoplasmosis shows no efficacy against Toxoplasma long-term chronic infections, resulting in the recrudescence of active infections. New drugs are imperatively needed. During mammalian cell invasion, the parasite creates a specialized parasitophorous vacuole (PV) that is demarcated from the host cytoplasm by a unique membrane containing parasite proteins. Toxoplasma is notorious for extensively modifying the host cell and rerouting host organelles to its PV, largely for purposes of nutrient acquisition. The parasite usually establishes its 'nest' near the host peri-Golgi microtubule-organizing center. This region is at the intersection of the endocytic and biosynthetic pathways, and PV positioning in this area of the cell could facilitate the interception of vesicular traffic and satisfy parasite requirements for nutrients and lipid membranes. In support of this hypothesis, we demonstrated that T. gondii has a marked requirement for host cell lipids derived both from lysosomes (e.g. cholesterol) and the Golgi (e.g. sphingolipids). The PV does not fuse with host organelles, and the mechanisms by which the parasite retrieves lipids from endocytic and exocytic organelles are still poorly understood. The overall goal of our proposal is to decipher the molecular details of host lysosome and Golgi exploitation by Toxoplasma. We previously showed that Toxoplasma uses the host microtubular network to redirect host lysosomes to its vacuole and sequester these organelles within PV membrane invaginations formed by microtubules. This strategy allows the parasite to have access to nutrients provided by organelles of the endocytic cascade. However, extensive gaps remain in our current model. Specific Aim 1 proposes experiments to better understand the intersection of T. gondii with the host endocytic pathway. We will clarify the nature and contribution of the endocytic structures intercepted by Toxoplasma to parasite development. We will examine the molecular mechanism of implicated in host microtubule-PV interaction and PV membrane transformations for lysosome sequestration. Almost nothing is known about the role of host Golgi during Toxoplasma infection. Our preliminary data show that the parasite associates with the Golgi, fragments this organelle into Golgi ministacks that align along the PV and intercepts the Golgi vesicular trafficking by engulfing secretory vesicles into the PV. This strategy may allow the parasite to retrieve Golgi lipids from sequestered Golgi vesicles. Specific Aim 2 combines experiments to verify our hypothetic model on host- Golgi-PV interaction. We will study the mechanisms leading to host Golgi breakdown and the delivery of Golgi vesicles to the PV by identifying the host Golgi proteins that are targeted by the parasite. We will explore the connection between Golgi remodeling and lipid salvage by the parasite. Our results may raise the provocative notion of novel treatments against Toxoplasma based on interference with host organelle functions.