Identifying human genes required by tick-borne pathogens

描述（由申请人提供）：嗜吞噬细胞无原体（Ap）及其宿主的生物学是相互交织的。数千年来共同进化的特定共同适应控制着疾病病原体与人类之间的相互作用。本提案将利用人类细胞和人类病原体在分子水平上解决寄生虫和宿主的共生问题。要了解Ap如何利用宿主细胞，需要了解宿主为微生物提供了什么。我们的中心假设是，Ap重新引导细胞代谢机制为自己的使用。我们假设Ap参与分子寄生以补充其流线型基因组，这在许多代谢途径中都是缺乏的。因此，我们建议通过使用短干扰RNA （siRNA）伪病毒文库系统地敲除47400个人类基因，然后转染具有特定siRNA结构的细胞，来确定人类宿主细胞在支持Ap寄生中的作用。沉默对Ap感染至关重要的细胞基因的表达将使细胞无法支持Ap。我们将使用siRNA文库进行基因组筛选，以确定人类细胞中Ap的粘附、侵袭和生长所需的宿主因子。可以分离出干扰Ap感染的基因敲低的宿主细胞，并利用人类基因组DNA阵列鉴定出相关的siRNA种类。为了评估得到的基因列表，我们将它们映射到京都基因与基因组百科全书（KEGG）途径数据库，将沉默的基因与代谢途径联系起来。在微阵列上鉴定的选定基因将使用基于pMaleficent睡美人转座子的RNAi传递系统进行靶向，以在人类宿主细胞系中产生特异性siRNA的遗传整合和表达。Ap进入宿主细胞并在宿主细胞中繁殖的能力将通过光学和荧光显微镜、延时显微镜、流式细胞仪分析和使用荧光板读取器进行评估。我们期望产生具有明确和稳定的基因敲低表型的人类细胞系。在KEGG数据库中重新定位受影响的基因将揭示并确认Ap对人类代谢物的特定依赖性，这可能导致针对这些和其他细胞内病原体的新治疗和疫苗。虽然人类基因组已经测序，但很大比例的orf功能未知。我们预计人类宿主细胞与这些高度特化的细胞内病原体的相互作用将揭示一些未知人类基因的本质。

DESCRIPTION (provided by applicant): The biology of Anaplasma phagocytophilum (Ap) and its host are intertwined. Specific co-adaptations which have co-evolved over millennia govern the interactions between disease agents and humans. This proposal will address the symbiosis of parasite and host at the molecular level using human cells and a human pathogen. To understand how Ap exploits its host cells requires an understanding of what the host provides for the microbes. Our central hypothesis is that Ap redirects the cellular metabolic machinery for its own use. We hypothesize that Ap engages in molecular parasitism to complement its streamlined genome, that is deficient in many metabolic pathways. Therefore, we propose to determine the role of human host cells in supporting Ap parasitism through systematic knockdown of 47,400 human genes using a pseudoviral library of short interfering RNA (siRNA), followed by transfeetion of cells with specific siRNA constructs. Silencing expression of cellular genes critical for Ap infection will render cells unable to support Ap. We will conduct a genomic screen using an siRNA library to identify host factors required for adhesion, invasion, and growth of Ap in human cells. Host cells with gene knockdown(s) that interfere with Ap infection can be isolated, and the siRNA species responsible identified using human genomic DNA arrays. To evaluate the resultant list of genes, we will map them to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database to link the silenced genes to metabolic pathways. Selected genes identified on microarrays will be targeted using the pMaleficent Sleeping Beauty transposon based RNAi delivery system to produce heritable integration and expression of specific siRNA in human host cell lines. The ability of Ap to enter and multiply in host cells will be assessed by light and fluorescent microscopy, time-lapse microscopy, FACS analysis and by using a fluorescent plate reader. We expect to generate human cell lines with defined and stable gene knockdown phenotypes. Remapping affected genes on the KEGG database will reveal and confirm specific dependencies of Ap on human metabolites that may lead to new treatments and vaccines against these and other intracellular pathogens. Although the human genome has been sequenced, a significant proportion of ORFs are of unknown function. We anticipate that the interaction of the human host cells with these highly specialized intracellular pathogens will shed light,on the nature of some unidentified human genes.