Comparison of Human Ehrlichiosis Agent Genomes

描述（由申请方提供）：查菲埃立克体感染单核细胞和巨噬细胞，并引起一种称为人单核细胞埃立克体病（HME）的潜在致命性新发传染病。完整的E.阿肯色州的查菲虫基因组序列于2006年发表。这也是唯一的菌株的实验发病机理数据是可用的。HME的严重程度从无症状感染到死亡不等。我们的总体假设是，比较基因组序列分析结合比较致病性研究的多个E。chaffeensis分离物可以为潜在的E.查菲菌毒力决定因子和新的干预目标。具体目标如下： 1.鉴定E.使用密集平铺的微阵列通过CGH检测查菲菌株，并通过DNA测序确认。 2.测定E.在建立的动物模型中， 3.分析宿主对E. chaffeensis菌株的不同毒力，并确认使用定量RT-PCR的结果，和抗体特异性选择的细胞因子和宿主信号分子。 4.对多态性E. 1）通过表达重组蛋白的毒性和无毒力形式或通过用候选毒性基因转染宿主细胞以研究它们对靶宿主细胞功能的影响; 2）通过制备重组蛋白的抗体并通过共聚焦免疫荧光或免疫金电子显微术定位蛋白：3）通过宿主/细菌蛋白复合物的免疫沉淀和酵母双杂交系统鉴定宿主相互作用蛋白; 4）通过测定抗体的体外感染中和作用;和/或通过用重组蛋白免疫有免疫活性的动物或用特异性抗体被动免疫SCID小鼠，并用强毒株攻击它们。该研究将鉴定新的E.查菲菌毒力决定因子及其致病机制结果可能指向潜在的化疗，化学预防和/或疫苗候选人治疗和预防人类埃立克体病。

DESCRIPTION (provided by applicant): Ehrlichia chaffeensis infects monocytes and macrophages, and causes a potentially fatal emerging infectious disease called Human monocytic ehrlichiosis (HME). The complete E. chaffeensis Arkansas genome sequence was published in 2006. This is also the only strain for which experimental pathogenesis data are available. Severity of HME varies from asymptomatic infection to death. Our overall hypothesis in the proposed study is that comparative genome sequence analysis combined with comparative pathogenesis studies of multiple E. chaffeensis isolates can provide valuable insights into potential E. chaffeensis virulence determinants and new intervention targets. The specific aims are as follows: 1. Identify polymorphic genes or genomic regions of E. chaffeensis strains by CGH using densely tiled microarray and confirm by DNA sequencing. 2. Determine phenotypes of E. chaffeensis strains in established animal models. 3. Analyze temporal transcriptome profiles of hosts in response to E. chaffeensis strains of distinct virulence, and confirm the results using quantitative RT-PCR, and antibodies specific to selected cytokines and host signaling molecules. 4. Functionally characterize polymorphic E. chaffeensis genes associated with virulence 1) by expressing the virulent and the avirulent versions of recombinant proteins or by transfection of host cells with the candidate virulent gene to study their effects on target host cell functions; 2) by making antibodies to the recombinant proteins and localizing the proteins by confocal immunofluorescent or immunogold electron microscopy: 3) by identifying host interacting proteins by immunoprecipitation of host/bacterial protein complexes and by yeast two-hybrid system: 4) by determining in vitro infection neutralizing effects of the antibodies; and/or by immunizing immunocompetent animals with recombinant proteins or passively immunizing SCID mice with specific antibodies, and challenging them with the virulent strain. The proposed study will identify novel E. chaffeensis virulence determinants and their pathogenic mechanisms. The results may point to potential chemotherapy, chemopreventive and/or vaccine candidates for treatment and prevention of human ehrlichiosis.