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

描述（由申请人提供）：近年来，埃立克体属和无形体属立克次体引起的疾病日益引起人类健康问题，是美国蜱媒感染的第二大原因。它们包括由E. chaffeensis。尽管脊椎动物和蜱类宿主有复杂的防御系统，立克次氏体还是逃避了宿主的清除。E.在巨噬细胞和蜱细胞中的chaffeensis在通过其分泌途径分泌的几种外膜蛋白和效应蛋白的表达上显著不同。我们报道了差异表达有助于不同的宿主反应和延迟清除宿主。我们资助的项目的中心假设是，E。Chaffeensis差异调节基因表达，宿主特异性基因表达对其在脊椎动物和蜱虫中的存活至关重要。该项目的具体目标是：1）建立用于定位埃里希启动子的体外转录系统，2）定位p28-Omp位点的两个差异表达基因的转录机制，3）评估巨噬细胞和蜱细胞环境对差异表达的贡献，以及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。目标4是最具挑战性的，因为我们是第一个开发在大肠杆菌中产生靶向和随机突变的方法的人。chaffeensis;我们在几个基因组位置制造了突变，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。获资助项目的进展构成了此次续期申请的坚实基础。具体目标是：1）表征E. chaffeensis RNA聚合酶复合物的研究，以支持对宿主特异性差异基因表达的理解; 2）通过鉴定为E. chaffeensis体内生长，和3）进行突变分析和体内筛选以鉴定E.在脊椎动物和蜱宿主中的chaffeensis发病机制。

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.