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式节肢动物(果蝇)系统中复制节肢动物(壁虱)传播的病原体查菲埃利希氏菌所必需的宿主基因。主要研究人员完成了在允许和不允许的条件下感染查菲埃希氏菌的果蝇S2细胞的基因表达阵列分析。这项分析揭示了528个基因在允许的细菌生长条件下显著上调(比未感染的对照组高1.5倍)。工作假设是，这些基因中的一个或多个是查菲埃希氏菌在宿主中最佳生长所必需的，这些基因的中断将影响细菌的生长。第一个具体目标将是感染携带我们阵列识别的基因突变的成年苍蝇。110个携带在允许条件下特别上调的基因突变的苍蝇品系可以作为具有存活能力的成虫获得。如果主要研究人员在这一筛选中发现了任何基因，他们将使用双链RNA来沉默该基因，以验证该果蝇基因的沉默将影响果蝇S2类血细胞中细菌的复制。第二个具体目标将是使用RNAi文库来识别在成年果蝇中没有以突变形式表达的宿主基因，这些基因对查菲埃希氏杆菌的细胞内生长也是必不可少的。在果蝇RNAi筛选中心(DrSc)进行全面的RNAi筛选将使我们能够研究S2细胞中基因下调是如何影响感染的。主要研究人员预计，他们将识别出多个基因，当这些基因被干扰时，将在体内(在果蝇中)和体外(在S2细胞中)导致较低水平的查菲埃希氏菌感染。这一结果将允许主要研究人员接受他们的实验假设，并允许他们对所需基因在单个或多个生化/细胞途径中是否必需进行分类。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，以确定在脊椎动物或无脊椎动物(例如扁虱)系统中用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确定查菲埃菲氏杆菌复制所需的宿主依赖因子，并将有助于确定用于预防人类单核细胞埃立克体病和其他立克次体疾病的翻译应用程序。 公共卫生相关性：这是一个与生物医学相关的项目，研究哪些宿主基因是成功复制作为人类C类病原体的细胞内细菌所必需的。

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.