Vector and Host Contributions to the Regulation of E. chaffeensis Gene Expression

描述（由申请人提供）：由埃利希氏体和无形体属立克次体引起的疾病是近年来日益严重的人类健康问题，是美国蜱传感染的第二大原因。它们包括由沙非埃希虫引起的人类单核细胞埃利希体病。尽管脊椎动物和蜱虫宿主有复杂的防御系统，立克次体还是逃避了宿主的清除。巨噬细胞和蜱虫细胞分泌的几种外膜蛋白和效应蛋白在巨噬细胞和蜱虫细胞中的表达存在显著差异。我们报道了差异表达有助于宿主的不同反应和延迟清除。我们资助的项目的中心假设是沙非沙螨对基因表达的差异调节，宿主特异性基因表达对其在脊椎动物和蜱虫细胞中的生存至关重要。该项目的具体目标是：1)建立埃利希启动子定位的体外转录系统；2)绘制p28-Omp位点两个差异表达基因的转录机制；3)评估巨噬细胞和蜱虫细胞环境对差异表达的贡献；4)开发评估敲除突变的方法，以评估宿主细胞特异性差异表达的生物学相关性。我们成功实现了目标1和目标2；虽然在目标3方面取得了一些进展，但我们需要在更新的项目中进一步扩大这一领域。目标4是最具挑战性的，因为我们是第一个开发出在沙非叶蝉中创建靶向和随机突变的方法；我们在几个基因组位置创造了突变，并提出了鉴定病原体体内生长所必需的基因的第一个证据。获资助项目的进展是本续期申请的坚实基础。具体目的是：1)表征查菲伊蚊RNA聚合酶复合物，以支持理解宿主特异性差异基因表达；2)通过表征查菲伊蚊在体内生长所必需的三个基因的突变，评估宿主特异性差异表达的意义；3)进行突变分析和体内筛选，以确定在脊椎动物和蜱虫宿主中查菲伊蚊发病所必需的其他基因。

DESCRIPTION (provided by applicant): Illnesses caused by rickettsiales of the genera Ehrlichia and Anaplasma are a growing human health concern in recent years and are the second leading cause of tick-borne infections in the USA. They include human monocytic ehrlichiosis caused by E. chaffeensis. Despite the sophisticated systems of defense in vertebrate and tick hosts, the rickettsiales evade the host clearance. E. chaffeensis in macrophages and tick cells differ significantly in expression of several outer membrane proteins and effector proteins secreted via its secretary pathways. We reported that differential expression contributes to the varied host response and delayed clearance in a host. The central hypothesis of our funded project is that E. chaffeensis differentially regulates gene expression and that the host-specific gene expression is essential for its survival in vertebrate and tick cels. Specific aims of the project are to 1) establish in vitro transcription system for mapping Ehrlichi promoters, 2) map transcriptional machinery of two differentially expressed genes of the p28-Omp locus, 3) evaluate the contributions of macrophage and tick cell environments for differential expression, and 4) develop methods to evaluate knockout mutations to assess the biological relevance of host cell specific differential expression. We successfully accomplished the goals of aims 1 and 2; although some progress was made in aim 3, we need to expand this area more in the renewed project. Aim 4 is the most challenging because we are the first to develop methods in creating both targeted and random mutations in E. chaffeensis; we created mutations in several genomic locations and presented the first evidence in identifying genes essential for the pathogen's in vivo growth. Progress from the funded project forms the strong foundation for this renewal application. Specific aims are 1) characterize E. chaffeensis RNA polymerase complex in support of understanding host-specific differential gene expression, 2) evaluate the significance of host-specific differential expression by characterizing mutations in three genes identified as essential for E. chaffeensis in vivo growth, and 3) perform mutational analysis and in vivo screening to identify additional genes essential for the E. chaffeensis pathogenesis in vertebrate and tick hosts.