Investigating hepatocyte signaling driven by host-pathogen interactions

描述(申请人提供)：我的职业目标是建立一个独立的研究小组，同时发现病原体操纵宿主细胞的方式，并为培训和激励下一代 科学家。我在细胞信号、技术开发、蛋白质/蛋白质相互作用和寄生虫学方面的背景使我能够应用各种强大的方法来解决关于病原体及其如何与宿主细胞相互作用的重要问题。我在斯特凡·卡普的团队从事博士后工作期间就开始了这些努力，主要研究疟疾寄生虫及其肝细胞宿主。我打算进一步扩展这些方法，以研究不同病原体之间的异同。获得K99/R00奖将极大地帮助完成建立一个富有成效和可持续发展的研究小组所需的重要任务，并使我能够充分实现我的目标，领导一个专注于宿主-寄生虫相互作用的基础生物学的小组。病原菌与寄主细胞之间的相互作用网络是复杂的，在促进病原菌存活和生命周期进程方面具有很强的适应性。在这个建议中，我们利用了两种相关的顶端复合体寄生虫：与肝细胞环境共同进化使其得以生存的疟原虫寄生虫，以及持续存在而不需要肝细胞感染的弓形虫寄生虫。我们最近已经证明，肝期疟疾寄生虫通过降低肿瘤抑制因子P53的水平来微妙地塑造它们的宿主细胞，并且从基因上或用小分子逆转这种扰动极大地降低了寄生虫在肝脏内发展的能力。在这项建议中，我们的目标是追求三个主要目标。首先，我们计划进一步阐明疟疾寄生虫依赖低宿主P53维持细胞内生存的机制，并调查这种干扰是否是弓形虫细胞内复制所必需的。然后，我们将调查哪些寄生虫分子与P53相互作用，以及这种相互作用是否对疟原虫肝脏阶段的发育至关重要。最后，我们建议通过使用蛋白质裂解物微阵列广泛询问肝细胞中响应啮齿动物和人类疟原虫以及弓形虫寄生虫的信号蛋白来扩展我们已经有趣的关于疟原虫感染的肝细胞的分子数据集，这种方法允许我们使用来自大约10,000个感染细胞的裂解物来监测数百种蛋白质和翻译后修饰水平。综上所述，这里提出的实验将有助于更深入地了解细胞内寄生虫繁衍的环境，并揭示预防药物可能针对的相互作用节点。

DESCRIPTION (provided by applicant): My career goal is to establish an independent research group, which simultaneously makes discoveries regarding the ways pathogens manipulate their host cells and contributes to the training and inspiring of the next generation of scientists. My background in cellular signaling, technology development, protein/protein interactions and parasitology has uniquely positioned me to apply a variety of powerful approaches to address important questions about pathogens and how they interact with their host cells. I have begun these efforts during my postdoctoral work in Stefan Kappe's group focusing on the malaria parasite and its hepatocyte host. I intend to expand these approaches moving forward to study the similarities and differences between divergent pathogens. Receipt of this K99/R00 award will greatly aid in accomplishing the vital tasks required to establish a productive and sustainable research group, and allow me to fully pursue my goal of leading a group focused on the fundamental biology of host-parasite interactions. Interaction networks of intracellular pathogens and their host cells are complex and predicted to be adaptive in promoting pathogen survival and life cycle progression. In this proposal, we utilize two related apicomplexan parasites: Plasmodium parasites whose co-evolution with the hepatocyte milieu has enabled their survival and Toxoplasma parasites whose persistence has occurred without requiring hepatocyte infection. We have recently demonstrated that liver stage malaria parasites subtly mold their host cells by lowering levels of the tumor suppressor p53, and that reversing this perturbation genetically or with small molecules dramatically reduces the ability of the parasite to develop within the liver. In this proposal, we aim to pursue three main objectives. First, we plan to further elucidate the mechanisms governing the malaria parasites dependence on low host p53 for intracellular survival, and investigate whether or not this perturbation is als required for robust Toxoplasma intracellular replication. We will then investigate which parasite molecules interact with p53 and whether or not this interaction is critical for Plasmodium liver stage development. Finally, we propose to expand upon our already intriguing molecular dataset for Plasmodium-infected hepatocytes by extensively interrogating signaling proteins in hepatocytes in response to both rodent and human Plasmodium parasites as well as Toxoplasma parasites using protein lysate microarrays, an approach that allows us to monitor hundreds of protein and post- translational modification levels using lysates derived from ~10,000 infected-cells. Taken together, the experiments proposed here will facilitate a deeper understanding of the environment in which intracellular parasites thrive and reveal interaction nodes that could be targeted by prophylactic drugs.