Comparison of Human Ehrlichiosis Agent Genomes

描述(申请人提供)：查菲埃立克体感染单核细胞和巨噬细胞，并导致一种潜在的致命的新出现的传染病，称为人类单核细胞埃立克体病(HME)。阿肯色州查菲肠杆菌的完整基因组序列于2006年公布。这也是唯一有实验发病机制数据的菌株。HME的严重程度从无症状感染到死亡不等。我们在这项研究中的总体假设是，比较基因组序列分析与多个查菲埃希菌分离株的致病机制比较研究相结合，可以为查菲埃希菌潜在的毒力决定因素和新的干预靶点提供有价值的见解。具体目标如下： 1.利用高密度芯片CGH技术鉴定查菲乳杆菌菌株的多态基因或基因组区域，并经DNA测序证实。 2.在已建立的动物模型中确定查菲埃希菌菌株的表型。 3.分析寄主对不同毒力的查菲埃希氏菌菌株的时间转录组图谱，并使用定量RT-PCR和针对选定的细胞因子和寄主信号分子的抗体来验证结果。 4)通过表达重组蛋白的毒力和无毒版本，或通过将候选毒力基因导入宿主细胞以研究它们对靶宿主细胞功能的影响；2)针对重组蛋白制造抗体，并通过共聚焦免疫荧光或免疫金电子显微镜定位蛋白：3)通过免疫沉淀宿主/细菌蛋白复合体和酵母双杂交系统鉴定与宿主相互作用的蛋白：4)通过确定抗体的体外感染中和作用；和/或通过用重组蛋白免疫具有免疫活性的动物或用特定的抗体被动免疫SCID小鼠，并用毒力株攻击它们。这项拟议的研究将确定新的查菲埃希氏菌毒力决定因素及其致病机制。这一结果可能为治疗和预防人类埃立克体病提供潜在的化疗、化学预防和/或疫苗候选。

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.