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

描述（由申请人提供）：由蜱传立克氏体和无形体属的立克氏体引起的疾病近年来已成为日益关注的健康问题。这些疾病包括人单核细胞埃利体病、人粒细胞埃利体病和人粒细胞无形体病，它们分别由沙菲埃利体、伊温伊氏埃利体和嗜吞噬细胞无形体引起。尽管节肢动物和脊椎动物复杂的细胞环境具有复杂的防御系统，立克次体进化出逃避宿主清除的策略。例如，沙非伊虫在巨噬细胞和蜱虫细胞中的形态差异很大，蛋白表达模式也不同。表达模式包括p28-Omp多基因座的差异表达，该多基因座包含22个基因。巨噬细胞中p28 Omp基因的表达主要局限于p28 Omp基因19，而蜱虫细胞中表达的抗原主要来自p28 Omp基因14。我们的研究还表明，在脊椎动物和蜱虫细胞中的差异表达有助于改变宿主的反应，例如，与巨噬细胞来源的细菌相比，蜱虫细胞来源的沙非埃希菌在脊椎动物宿主中的清除延迟。本研究将着重从p28-Omp多基因位点对基因表达调控的基本认识。本研究的中心假设是沙非沙蚤p28-Omp位点的转录在响应环境信号时受到差异调节，宿主细胞特异性基因表达对于病原体在脊椎动物和蜱虫细胞环境中的生存至关重要。本研究的具体目的是；1)建立体外转录和翻译系统定位埃利希体启动子；2)定位p28-Omp基因座差异表达基因14和19的转录机制；3)评估巨噬细胞和蜱虫细胞环境对基因14和19差异表达的贡献；4)评估p28-Omp基因14和19的敲除突变，以评估宿主细胞特异性表达的生物学相关性。本研究结果将为了解沙非叶蝉基因调控以及巨噬细胞和蜱虫细胞中立克次体对差异表达蛋白表达缺失的反应提供重要信息。

DESCRIPTION (provided by applicant): Illnesses caused by the tick-borne rickettsiales of the genera Ehrlichia and Anaplasma have been a growing health concern in recent years. These illnesses include human monocytic ehrlichiosis, human granulocytic ehrlichiosis and human granulocytic anaplasmosis which are caused by Ehrlichia chaffeensis, E. ewingii and Anaplasma phagocytophilum, respectively. Despite the complex cellular environment of arthropods and vertebrates having sophisticated systems of defense, the rickettsiales evolved strategies to evade host clearance. For example, E. chaffeensis in macrophages and tick cells differ significantly in their morphology with distinct patterns of protein expression. The expression patterns include the differential expression from the p28-Omp multigene locus which contains 22 genes. The p28 Omp gene expression is restricted primarily to the p28-Omp gene 19 in macrophages, whereas in tick cells the expressed antigen is predominantly from the p28-Omp gene 14. Our studies also demonstrate that the differential expression in vertebrate and tick cells contributes to the altered host response, such as the delayed clearance in a vertebrate host for tick cell- derived E. chaffeensis compared to that for macrophage-derived bacteria. This study will focus on the basic understanding of the regulation of gene expression from the p28-Omp multigene locus. The central hypothesis of this study is that the transcription from the p28-Omp locus in E. chaffeensis is differentially regulated in response to environmental signals and that the host cell-specific gene expression is essential for pathogen's survival in vertebrate and tick cell environments. Specific aims of this study are; 1) establish in vitro transcription and translation system for mapping Ehrlichia promoters, 2) map transcriptional machinery in differentially expressed genes 14 and 19 of the p28-Omp locus, 3) evaluate the contributions of macrophage and tick cell environments for differential expression from genes 14 and 19, and 4) evaluate knockout mutations for the p28-Omp genes 14 and 19 to assess the biological relevance of host cell specific expression. The results from this study will provide important information for understanding E. chaffeensis gene regulation and how the rickettsiale in macrophages and tick cells respond to the loss of expression from a differentially expressed protein.