Definition of host genes required for intracellular pathogen growth

描述（由申请人提供）：该项目的主要目标是确定在模型节肢动物（果蝇）系统中复制节肢动物（蜱虫）传播的病原体沙菲埃利希体所需的宿主基因。主要研究人员完成了在允许和非允许条件下感染沙非叶蝉的果蝇S2细胞的基因表达阵列分析。该分析显示，在允许细菌生长的条件下，528个基因明显上调（比未感染的对照组高1.5倍）。目前的假设是，这些基因中的一个或多个是沙费梭菌在宿主体内最佳生长所必需的，而这些基因的破坏将影响细菌的生长。第一个具体目标将是感染携带我们的阵列鉴定的基因突变的成年苍蝇。110种携带基因突变的蝇系，在适宜条件下特异性上调，可作为可育成虫获得。如果主要研究人员在这个筛选中发现任何基因，他们将使用双链RNA来沉默基因，以验证沉默果蝇基因会影响果蝇S2血细胞样细胞中细菌的复制。第二个具体目标将是使用RNAi文库来鉴定在成年果蝇中未以突变形式表达的宿主基因，这些基因也是沙非吉虫细胞内生长所必需的。果蝇RNAi筛选中心（The Drosophila RNAi Screening Center， DRSC）全面的RNAi筛选将使我们能够研究S2细胞中基因的下调是如何影响感染的。主要研究人员预计，他们将鉴定出多个基因，当这些基因被破坏时，它们将在体内（在苍蝇中）和体外（在S2细胞中）引起较低水平的沙非伊虫感染。这一结果将允许主要研究人员接受他们的实验假设，并允许他们分类所需的基因在单个或多个生化/细胞途径中是必需的。从这项工作中获得的数据将使主要研究人员能够专注于已识别的基因，以确定脊椎动物或无脊椎动物（例如蜱虫）系统中的药物操纵生化途径如何影响细菌生长。因此，该项目提出了一种高度创新的方法来鉴定沙菲埃希体复制所需的宿主依赖因子，并将有助于确定预防人类单核细胞埃利希体病和其他立克次体疾病的转化应用。

DESCRIPTION (provided by applicant): The major goal of this project is to define host genes necessary for the replication of an arthropod (tick)-transmitted pathogen, Ehrlichia chaffeensis, in a model arthropod (Drosophila) system. The principal investigators completed a gene expression array analysis of Drosophila S2 cells infected with E. chaffeensis under permissive and non-permissive conditions. This analysis revealed 528 genes that were significantly upregulated (>1.5 fold higher than uninfected control) exclusively during permissive bacterial growth conditions. The working hypothesis is that one or more of these genes are necessary for optimal growth of E. chaffeensis in a host and disruption of those genes will affect bacterial growth. The first specific aim will be to infect adult flies carrying mutations in genes identified by our array. 110 fly lines that carry mutations in genes specifically upregulated during permissive conditions can be obtained as viable-fertile adults. Should the principal investigators identify any genes in this screen, they will use double stranded RNA to silence the gene to validate that silencing of that Drosophila gene will affect the replication of bacteria in Drosophila S2 hemocyte-like cells. The second specific aim will be use an RNAi library to identify host genes that are not expressed in mutant form in adult flies that are also essential for intracellular growth of E. chaffeensis. The availability of comprehensive RNAi screen at the The Drosophila RNAi Screening Center (DRSC) will allow us to investigate how down regulation of genes in S2 cells affects infection. The principal investigators anticipate that they will identify multiple genes that when disrupted will cause lower levels of infection by E. chaffeensis in vivo (in flies) and in vitro (in S2 cells). That outcome will allow the principal investigators to accept their experimental hypothesis and allow them to sort whether the required genes are necessary in a single or multiple biochemical/cellular pathways. The data obtained from this work will allow the principal investigators to focus on the identified genes to determine how manipulation of biochemical pathways with drugs in vertebrate or invertebrate (e.g. tick) systems affect bacterial growth. Therefore, this project presents a highly innovative way to identify host-dependent factors necessary for E. chaffeensis replication and will be useful for identifying translational applications to prevent human monocytic ehrlichiosis and perhaps other Rickettsial diseases. PUBLIC HEALTH RELEVANCE: This is a biomedically related project that addresses which host genes are necessary for the successful replication of an intracellular bacteria that is a human category C pathogen.