Profiling the Leishmania-macrophage Host-pathogen Infectome

描述（由申请人提供）： 病原体中基因表达的分析和宿主-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，我们专注于原生动物寄生虫利什曼原虫，人类皮肤利什曼病的病原体。虽然在过去的20年里已经取得了重大进展，了解细胞内生存利什曼原虫的分子和细胞基础，我们仍然缺乏有关细胞内无鞭毛体的生物学信息。特别是，很少有人知道的基因表达的寄生虫之前和之后的吞噬溶酶体融合和所需的基因，以促进适应，并在宿主细胞内生存。此外，在感染的背景下，在人巨噬细胞中独特激活和表达的基因尚未系统地确定。最近出现的下一代测序技术开辟了前所未有的机会，以获得一个深入了解转录组的L。主要的和巨噬细胞在感染的情况下。本研究的目的是获得第一个全面的转录组分析数据的三个主要发展阶段的L。major（目的1），同时分析L.主要沿着受感染的人宿主细胞巨噬细胞的转录应答（目的2）。这些研究应该提供深入了解宿主防御机制启动的巨噬细胞，并导致更好地了解什么是巨噬细胞“看到”的寄生虫，因为它被摄入。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（Aim 3）确定重要的利什曼原虫-人类蛋白质相互作用（PPI）伙伴的子集。很少有实验研究调查了宿主-病原体PPI。将其扩展到细胞内寄生虫利什曼原虫，不仅可以识别使该寄生虫能够感染其宿主细胞、获得营养并逃避其免疫防御的PPI，而且还将提供对一般发病机制的更全面的功能观点。总之，这项研究将提供对细胞内原生动物寄生虫及其巨噬细胞宿主细胞的转录组的第一次大规模和同时的询问，并将提供对感染组的第一次观察，感染组是宿主细胞基因组和蛋白质组的一部分，对病原体的感染很重要，以及病原体基因组/蛋白质组的一部分，允许它破坏一些宿主细胞受体的功能，信号蛋白和分子机制。.

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. .