Host cell manipulation by Toxoplasma

描述（由申请人提供）：专性细胞内原生动物刚地弓形虫是弓形体脑炎的病原体，弓形体脑炎仍然是艾滋病患者死亡的主要原因。目前可用的弓形虫病治疗方法对弓形虫长期慢性感染无效，导致活动性感染复发。迫切需要新的药物。在哺乳动物细胞入侵过程中，寄生虫产生一个专门的寄生虫空泡（PV），通过含有寄生虫蛋白的独特膜与宿主细胞质区分开来。弓形虫是臭名昭著的广泛修改宿主细胞和主机的细胞器改道到其PV，主要是为了获取营养的目的。寄生虫通常在宿主高尔基体周围微管组织中心附近建立其“巢”。该区域是在交叉口的内吞和生物合成途径，PV定位在这一领域的细胞可以促进拦截囊泡交通和满足寄生虫的营养物质和脂质膜的要求。为了支持这一假设，我们证明了T。弓形虫对来自溶酶体（例如胆固醇）和高尔基体（例如鞘脂）的宿主细胞脂质具有显著的需求。PV不与宿主细胞器融合，并且寄生虫从内吞和外吞细胞器检索脂质的机制仍然知之甚少。我们的总体目标是破译宿主溶酶体和高尔基体利用弓形虫的分子细节。我们以前表明，弓形虫使用的主机微管网络重定向主机溶酶体到其空泡和隔离PV膜内的微管形成的内陷这些细胞器。这种策略允许寄生虫获得由内吞级联的细胞器提供的营养。然而，在我们目前的模式中仍然存在着广泛的差距。具体目标1提出了实验，以更好地理解T的交集。弓形虫与宿主内吞途径。我们将澄清的性质和贡献的内吞结构拦截弓形虫寄生虫的发展。我们将研究宿主微管-PV相互作用和PV膜转化的分子机制。目前对宿主高尔基体在弓形虫感染过程中的作用还知之甚少。我们的初步数据表明，寄生虫与高尔基体，碎片这个细胞器成高尔基体小栈对齐沿着PV和拦截高尔基囊泡运输吞噬分泌囊泡进入PV。这种策略可能允许寄生虫从隔离的高尔基体囊泡中取回高尔基体脂质。具体目标2结合实验来验证我们关于宿主-高尔基体-PV相互作用的假设模型。我们将通过鉴定寄生虫靶向的宿主高尔基体蛋白来研究导致宿主高尔基体分解和高尔基体囊泡向PV递送的机制。我们将探讨高尔基体重塑和脂质救助之间的联系的寄生虫。我们的研究结果可能会提出一个挑衅性的概念，新的治疗方法对弓形虫的基础上干扰宿主细胞器的功能。 公共卫生相关性：人类寄生虫弓形虫是艾滋病毒/艾滋病患者脑部病变的最常见原因。这种病原体侵入哺乳动物细胞，并在一个专门的小生境内发育，该小生境作为调节宿主细胞功能的平台。我们的建议侧重于识别寄生虫开发的宿主细胞器中脂质摄取的独特功能，以发现新疗法的靶点。

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. PUBLIC HEALTH RELEVANCE: The human parasite Toxoplasma is the most common cause of brain lesions in HIV/AIDS patients. This pathogen invades mammalian cells and develops within a specialized niche that serves as a platform for modulation of host cell functions. Our proposal focuses on the identification of unique features of lipid uptake from host organelles developed by the parasite in order to uncover targets for new therapeutics.