Perturbations of host cell signaling by a complex hepatotropic pathogen

描述（由申请人提供）：细胞内病原体及其宿主细胞的相互作用网络是复杂的，预计在促进病原体存活方面具有适应性。在疟原虫肝细胞内肝阶段感染期间，疟原虫通过以下方式保护其宿主肝细胞： 防止其死亡并充分利用宿主细胞资源进行生长和复制。主机 肝细胞分子信号转导景观，巩固成功的肝阶段复制尚未阐明，但它是高度医学相关的。我们将使用在肝细胞感染后不同时间点停止的野生型和减毒寄生虫株作为工具，以阐明健康的野生型寄生虫如何操纵其宿主细胞与减毒寄生虫如何之间的差异，减毒寄生虫在停止后不久从肝脏中清除，无法做到这一点。在这个提议中，我们提出了充分描述寄生虫可以逃避的促凋亡环境的实验，以及什么样的内在和外在扰动导致甚至野生型寄生虫的死亡。接下来，我们将通过使用蛋白质裂解物微阵列广泛询问肝细胞中响应野生型和减毒寄生虫的信号蛋白来扩展我们已经有趣的分子数据集，这种方法使我们能够使用来自约10，000个肝脏阶段感染肝细胞的裂解物监测数百种蛋白质和翻译后修饰水平。我们建议监测肝细胞信号，不仅在啮齿动物疟疾感染，而且在响应最致命的人类疟疾寄生虫，恶性疟原虫，使用感染的肝细胞从小鼠与人源化肝脏。在初步研究中，我们最近将肿瘤抑制基因p53的下调与疟原虫肝脏期成功感染联系起来，并证明人为提高p53水平可以消除肝脏期寄生虫。我们计划进一步阐明寄生虫依赖低宿主p53生存的机制。最后，我们调查的作用，BH3结构域的蛋白质在肝细胞线粒体中发挥疟疾寄生虫肝脏阶段感染，并询问他们是否有针对性的寄生虫，以防止宿主细胞凋亡。拟议的研究将导致更全面地了解肝细胞信号转导景观，这对调节宿主对疟原虫肝脏阶段寄生虫感染的反应至关重要，包括对感染人类和影响人类健康的寄生虫的无与伦比的了解。实现我们的目标打开了用小分子改变肝细胞信号转导景观的可能性，这可以防止野生型寄生虫进展为有症状的红细胞感染。这种基于宿主的预防方法是新颖的，将避免对标准抗疟药物不断产生耐药性的巨大问题。这种方法进一步促进了许多肝细胞蛋白质已经是已知治疗抑制剂的靶标的事实。复杂的细胞内病原体对肝细胞信号传导的扰动也可能揭示肝细胞内信号传导系统的新的内在特征。

DESCRIPTION (provided by applicant): Interaction networks of intracellular pathogens and their host cells are complex and predicted to be adaptive in promoting pathogen survival. During malaria parasite intrahepatocytic liver stage infection, parasites protect their host hepatocyte by preventing its death and fully exploit the host cell resources for growth and replication. The host hepatocyte molecular signaling landscape that undergirds successful liver stage replication has not been elucidated, yet it is highly medically relevant. We will use wild-type and attenuated parasite strains that arrest at different points after hepatocyte infection as a tool to investigat the differences between how healthy, wild-type parasites manipulate their host cell and how attenuated parasites, which are cleared from the liver shortly after arrest, are unable to do so. I this proposal, we propose experiments that fully delineate the pro- apoptotic milieu that the parasite can evade, and what intrinsic and extrinsic perturbations lead to the demise of even a wild-type parasite. Next, we will expand upon our already intriguing molecular dataset by extensively interrogating signaling proteins in hepatocytes in response to both wild-type and attenuated 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 liver stage-infected hepatocytes. We propose monitoring hepatocyte signals not only in response to rodent malaria infection, but also in response to the most deadly human malaria parasite, Plasmodium falciparum, using infected hepatocytes from a mouse with a humanized liver. In preliminary studies, we have recently correlated the down-regulation of the tumor suppressor gene p53 with successful Plasmodium liver stage infection, and have demonstrated that artificially increasing p53 levels can eliminate liver stage parasites. We plan to further elucidate the mechanism behind the parasite's dependence on low host p53 for survival. Finally, we investigate the role that BH3-domain containing proteins in the mitochondria of hepatocytes play in malaria parasite liver-stage infection and ask if they are targeted by the parasite to prevent host cell apoptosis. The proposed studies will lead to a more comprehensive understanding of the hepatocyte signaling landscape critical to modulating the host response to Plasmodium liver stage parasite infection, including unparalleled understanding of parasites that infect humans and that impact human health. Accomplishing our aims opens the possibility of altering the hepatocyte signaling landscape with small-molecules that could prevent a wild-type parasite from progressing to symptomatic erythrocyte infection. Such a host-based approach for prophylaxis is novel and will circumvent the massive problem of continuously developing resistance to standard antimalarial drugs. This approach is further fostered by the fact that many hepatocyte proteins are already targets of known therapeutic inhibitors. Perturbations of hepatocyte signaling by a complex intracellular pathogen might also reveal new intrinsic features of the signaling system within hepatocytes.