Gene Expression and Manipulation of Coxiella Burnetii

伯氏柯克斯体是Q热的病原体，是潜在的生物武器和选择剂，以及 已知的最具传染性的病原体之一。然而，关于这一义务的报道却很少。 细胞内因子的分子发病机制或发育周期，以及缺乏定位的系统 基因操作严重阻碍了研究进展。为了解决这种信息匮乏的问题，并 针对分子生物学技术的现状，我们提出：1)检测基因表达谱 小细胞变异体(SCV)和大细胞变异体(LCV)的发育周期，以识别基因和 允许柯克斯体在环境中长时间存活的蛋白质(SCV阶段)和 随后在宿主细胞吞噬小体的不适宜居住的范围内生长(LCV阶段)；2)分析和 比较体外和体内培养的柯克斯体和I期与II期的表达谱 生物体以确定潜在的毒力决定因素，以及3)开发一种定点突变系统 对于这种有机体来说，允许进行常规的基因操作和未来的减毒 疫苗株。这些数据有望提供有关柯克斯体生活史的宝贵信息 各自涉及的基因和蛋白质，并应导致干扰细胞发育的策略 控制Q热。最后，柯克斯体的常规遗传操作系统是必不可少的分子 用于检查疑似毒力基因并在体内执行分子科赫假设的生物学工具。 项目互动-项目3(Harmsen)将与项目1(本研究)互动，因为它与活动相关 小鼠模型中的柯克斯氏菌。项目4(Pascual)和项目2(Jutila)将受益于项目1的数据 在阶段特定的基因产物上；假设SCV特定的基因/抗原参与 传播/早期感染和LCV特异性基因/抗原参与了细胞内复制。

Introduction- Coxiella burnetii is the etiologic agent of Q fever, a potential bioweapon and select agent, and one of the most infectious pathogens known. However, there are few reports concerning this obligate intracellular agent's molecular pathogenesis or developmental cycle, and lack of a system for site-directed genetic manipulation has severely hampered research progress. To address this dearth of information and to improve the current state of molecular biology technology, we propose to: 1) examine the expression profiles of small cell variants (SCVs) and large cell variants (LCVs) of the developmental cycle to identify genes and proteins that allow Coxiella to survive for extended periods of time in the environment (SCV stage) and to subsequently grow in the inhospitable confines of the host cell phagolysosome (LCV stage); 2) analyze and compare expression profiles of in vitro versus in vivo-cultivated Coxiella and phase I versus phase II organisms to identify potential virulence determinants, and 3) develop a system of site-directed mutagenesis for this organism to allow for the routine genetic manipulation and future development of an attenuated vaccine strain. These data are expected to provide valuable information on the life cycle of Coxiella and the respective genes and proteins involved, and should lead to strategies for disrupting cellular development to control Q fever. Finally, a system for routine genetic manipulation of Coxiella is an essential molecular biology tool for examining suspected virulence genes and performing molecular Koch's postulates in vivo. Project Interactions- Project 3 (Harmsen) will interact with Project 1 (this study) as it relates to in vivocultivated Coxiella from the mouse model. Projects 4 (Pascual) and 2 (Jutila) will benefit from Project 1 data on stage-specific gene products; SCV-specific genes / antigens are hypothesized to be involved in transmission / early infection and LCV-specific genes / antigens are involved in intracellular replication.