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节肢动物（蜱）传播的病原体查菲埃里希体在模型节肢动物（果蝇）系统中复制所需的宿主基因。主要研究人员完成了在许可和非许可条件下感染查菲埃里希氏体的果蝇 S2 细胞的基因表达阵列分析。该分析揭示了 528 个基因仅在允许的细菌生长条件下显着上调（比未感染对照高 1.5 倍以上）。工作假设是，这些基因中的一个或多个对于查菲埃里希菌在宿主中的最佳生长是必需的，并且这些基因的破坏将影响细菌的生长。第一个具体目标是感染携带我们阵列识别的基因突变的成年果蝇。 110 个携带在许可条件下特别上调的基因突变的果蝇品系可以获得可育的成虫。如果主要研究人员在此筛选中发现任何基因，他们将使用双链 RNA 沉默该基因，以验证果蝇基因的沉默是否会影响果蝇 S2 血细胞样细胞中细菌的复制。第二个具体目标是使用 RNAi 文库来鉴定在成年果蝇中不以突变形式表达的宿主基因，这些基因对于查菲埃里希体的细胞内生长也至关重要。果蝇 RNAi 筛查中心 (DRSC) 提供全面的 RNAi 筛查，这将使我们能够研究 S2 细胞中基因的下调如何影响感染。主要研究人员预计他们将鉴定出多个基因，这些基因被破坏后会导致体内（果蝇）和体外（S2 细胞）查菲埃里希体感染水平降低。这一结果将使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是否在单个或多个生化/细胞途径中是必需的进行分类。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（例如蜱）系统中使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识别查菲埃立克体复制所需的宿主依赖性因子，并将有助于确定预防人类单核细胞埃立克体病和其他立克次体疾病的转化应用。 公共卫生相关性：这是一个与生物医学相关的项目，旨在解决哪些宿主基因对于人类 C 类病原体的细胞内细菌的成功复制是必需的。

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.