Perturbations of host cell signaling by a complex hepatotropic pathogen

描述(由申请人提供)：细胞内病原体和它们的宿主细胞的相互作用网络是复杂的，并预测在促进病原体生存方面具有适应性。在疟疾寄生虫肝细胞内感染期间，寄生虫通过以下方式保护宿主肝细胞 防止其死亡，并充分利用宿主细胞资源进行生长和复制。东道主 支持成功的肝期复制的肝细胞分子信号格局尚未阐明，但它在医学上是高度相关的。我们将使用在肝细胞感染后不同时间点停滞的野生型和减毒寄生虫菌株作为工具，研究健康的野生型寄生虫如何操纵宿主细胞，以及减毒寄生虫是如何操作宿主细胞的，而减毒寄生虫在被捕后不久就被从肝脏清除，无法做到这一点。在这个提议中，我们提议进行实验，充分描绘寄生虫可以逃避的促凋亡环境，以及是什么内在和外在的扰动导致即使是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耐人寻味的分子数据集，这种方法允许我们使用来自大约10,000个肝脏阶段感染的肝细胞的裂解物来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，不仅是对啮齿动物疟疾感染的反应，也是对最致命的人类疟疾寄生虫恶性疟原虫的反应，使用带有人源化肝脏的小鼠的感染肝细胞。在初步研究中，我们最近将抑癌基因P53的下调与成功的肝期疟原虫感染联系起来，并证明了人为提高P53水平可以消除肝期寄生虫。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，我们研究了肝细胞线粒体中含有BH3结构域的蛋白在疟疾寄生虫肝期感染中所起的作用，并询问它们是否是寄生虫的靶标，以防止宿主细胞的凋亡。拟议的研究将导致对肝细胞信号转导格局的更全面的了解，包括对感染人类和影响人类健康的寄生虫的无与伦比的理解，这些信号对调节宿主对肝期疟原虫感染的反应至关重要。我们的目标的实现开启了用小分子改变肝细胞信号格局的可能性，这种小分子可以防止野生型寄生虫发展为有症状的红细胞感染。这种以宿主为基础的预防方法是新颖的，将绕过标准抗疟疾药物持续产生耐药性的巨大问题。许多肝细胞蛋白已经是已知治疗抑制剂的靶点，这一事实进一步促进了这种方法的发展。一种复杂的细胞内病原体对肝细胞信号的干扰也可能揭示肝细胞内信号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.