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

描述(由申请人提供)：近年来，埃利希菌属和无浆体属的壁虱传播立克次体引起的疾病日益成为人们关注的健康问题。这些疾病包括人单核细胞埃立克体病、人粒细胞埃立克体病和人粒细胞无形体病，分别由查菲埃立克体病、埃文吉氏埃希氏菌和吞噬细胞无形体引起。尽管节肢动物和脊椎动物拥有复杂的防御系统，但它们进化出了逃避宿主清除的策略。例如，巨噬细胞和扁虱细胞中的查菲埃希氏菌在形态上有很大的不同，具有不同的蛋白质表达模式。表达模式包括与p28-OMP多基因座的差异表达，该多基因座包含22个基因。P28OMP基因在巨噬细胞中的表达主要限于p28-OMP基因19，而在Tick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，脊椎动物和Tick细胞中的差异表达有助于宿主反应的改变，例如与巨噬细胞来源的细菌相比，Tick细胞来源的E.chaffesis在脊椎动物宿主中的清除延迟。本研究将集中于对p28-OMP多基因座基因表达调控的基本认识。本研究的中心假设是，查菲埃菲斯体内p28-OMP基因的转录受环境信号的差异调控，宿主细胞特异性基因的表达对病原体在脊椎动物和壁虱细胞环境中的生存是必不可少的。本研究的具体目的是：1)建立埃利希菌启动子的体外转录和翻译系统；2)定位p28-OMP基因的差异表达基因14和19的转录机制；3)评估巨噬细胞和扁虱细胞环境对基因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。这项研究的结果将为理解查菲埃希氏菌的基因调控以及巨噬细胞和扁虱细胞中立克次体如何应对差异表达蛋白的表达缺失提供重要信息。

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.