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

描述(申请人提供)：由埃利希菌属和无浆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，也是美国扁虱传播感染的第二大原因。其中包括由查菲埃希氏杆菌引起的人类单核细胞埃立克体病。尽管脊椎动物和壁虱的宿主有复杂的防御系统，但立克次体却逃避了宿主的清除。查菲埃希氏菌在巨噬细胞和壁虱细胞中的几种外膜蛋白和通过其分泌途径分泌的效应蛋白的表达存在显著差异。我们报道了差异表达导致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查菲埃希氏菌对基因表达有不同的调控，宿主特异性基因表达对其在脊椎动物和壁虱中的生存至关重要。该项目的具体目标是：1)建立体外转录系统，用于定位埃利奇启动子；2)定位p28-OMP基因座两个差异表达基因的转录机制；3)评估巨噬细胞和扁虱细胞环境对差异表达的贡献；4)建立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。我们成功地实现了目标1和目标2的目标；虽然目标3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。AIM 4是最具挑战性的，因为我们是第一个开发出在查菲肠杆菌中创建靶向和随机突变的方法；我们在几个基因组位置创建了突变，并提供了第一个证据来确定病原体在体内生长所必需的基因。从资助项目中取得的进展构成了这次续签申请的坚实基础。具体目标是1)鉴定查菲埃希氏菌RNA聚合酶复合体以支持理解宿主特异性差异基因表达，2)通过鉴定三个被确认为查菲埃希氏菌在体内生长所必需的基因的突变来评估宿主特异性差异表达的意义，以及3)进行突变分析和体内筛选，以确定在脊椎动物和壁虱宿主中查菲埃希氏菌致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.