Profiling the Leishmania-macrophage Host-pathogen Infectome

描述(由申请人提供)： 病原体基因表达的概要分析和宿主-病原体相互作用的研究是推动新药和疫苗发现项目的关键步骤。在这项研究中，我们重点关注原虫寄生虫大利什曼原虫，人类皮肤利什曼病的病原体。虽然在过去的20年里，在了解利什曼原虫细胞内存活的分子和细胞基础方面取得了重大进展，但我们仍然缺乏关于细胞内无鞭毛体生物学的信息。特别是，对寄生虫在吞噬-溶酶体融合之前和之后的基因表达以及促进宿主细胞适应和存活所需的基因知之甚少。此外，在人类巨噬细胞中唯一激活和表达的基因，在感染的背景下，还没有得到系统的确定。最近出现的下一代测序技术为深入了解主要乳杆菌的转录组和感染背景下的巨噬细胞打开了前所未有的机会。本研究的目的是获得第一个完整的主要发育阶段的转录组数据(目标1)，并同时分析细胞内无鞭毛体阶段的基因表达程序以及受感染的人类宿主细胞巨噬细胞的转录反应(目标2)。这些研究应该提供对巨噬细胞启动的宿主防御机制的洞察，并导致更好地理解巨噬细胞在寄生虫被吞噬时在其身上“看到”了什么。为了进一步研究寄生虫和人类巨噬细胞之间的相互作用，我们将使用成熟的酵母双杂交筛选法(Aim 3)鉴定重要的利什曼原虫-人类蛋白质相互作用(PPI)伙伴的子集。很少有实验研究研究寄主-病原体PPI。将这些扩展到细胞内寄生虫利什曼原虫，不仅可以识别PPI，使这种寄生虫能够感染宿主细胞，获得营养物质并逃避其免疫防御，而且还将提供更全面的致病机制的全球功能观点。总之，这项研究将提供第一次大规模和同时询问细胞内原生动物寄生虫及其巨噬细胞宿主细胞的转录本，并将提供对感染组的初步了解，感染组是宿主细胞基因组和蛋白质组中对病原体感染至关重要的部分，以及病原体基因组/蛋白质组中允许其颠覆某些宿主细胞受体、信号蛋白和分子机制功能的部分。。

DESCRIPTION (provided by applicant): Summary Analysis of gene expression in pathogens and investigation of host-pathogen interactions are a critical step in fueling new drug and vaccine discovery projects. In this study, we focus on the protozoan parasite Leishmania major, the causative agent of human cutaneous leishmaniasis. While significant progress has been made over the past 20 years toward understanding the molecular and cellular basis for intracellular survival of Leishmania, we still lack information regarding the biology of intracellular amastigotes. In particular, little is known about gene expression in the parasite prior to and following phago-lysosomal fusion and the genes required to facilitate adaptation to, and survival within, the host cell. Furthermore, genes that are uniquely activated and expressed in the human macrophage, in the context the infection, have not been systematically determined. The recent emergence of next-generation sequencing technologies opens up unprecedented opportunities to gain an in-depth view of the transcriptome of L. major and the macrophage in the context of infection. The goal of this study is to obtain the first comprehensive transcriptome profiling data for the three main developmental stages of L. major (Aim 1) and to simultaneously analyze gene expression programs in the intracellular amastigote stage of L. major along with the trancriptional response of the infected human host cell, the macrophage (Aim 2). These studies should provide insight into host defense mechanisms initiated by macrophages and lead to a better understanding of what the macrophage "sees" on the parasite as it is being ingested. To further examine the interactions between the parasite and the human macrophage, we will identify a subset of important Leishmania-human protein-protein interaction (PPI) partners using well-established yeast two-hybrid screens (Aim 3). Very few experimental studies have investigated host-pathogen PPIs. Extending those to the intracellular parasite Leishmania will not only allow the identification PPIs that enable this parasite to infect its host cells, acquire nutrients and evade its immune defenses, but will also provide a more global functional view of pathogenesis in general. In summary, this study will provide the first massive-scale and simultaneous interrogation of the transcriptomes of an intracellular protozoan parasite and its macrophage host cell and will provide a first look at the infectome, the part of a host cell's genome and proteome that is important for infection by a pathogen as well as the part of the pathogen's genome/proteome that allows it to subvert the functions of some host cell receptors, signaling proteins and molecular machinery. .