Identification of Coxiella burnetii Virulence Factors by Transposon Mutagenesis

描述(申请人提供)：伯氏柯克斯体是一种革兰氏阴性专性胞内细菌，可引起人类急性Q热和慢性感染。Q热研究人员面临的两个主要限制是1)缺乏遗传工具，2)细胞内培养的强制性要求。最近的进展开始解决这些限制：a)使用微需氧条件的生长介质，允许在宿主细胞外复制；b)有效克隆分离的新技术；c)基于Himar1的功能性转座子突变系统，以产生随机突变；d)转化稳定的穿梭载体质粒。本研究的目的是建立伯氏卡氏杆菌克隆的突变文库，并使用伯氏卡氏杆菌Nine Mile II期(RSA439，克隆4；降低毒力，BL2包容)作为未来研究完全毒力生物的模型，通过以下方式建立伯氏卡氏杆菌突变文库：1)使用改良的分离克隆，通过显微操作、荧光激活细胞分选(FACS)并在琼脂平板上增殖，建立伯氏卡氏杆菌的转座子突变克隆库。每种策略都将得到优化，并有助于正在进行的克隆文库组装。突变株将通过全基因组扩增(WGA)、Southern杂交、聚合酶链式反应(PCR)和救助性克隆对插入位置进行遗传学表征。2)确定其在体外致病过程中的作用。来自培养基和L929成纤维细胞感染系统的克隆将与野生型在培养基和几个组织培养模型细胞中的生长速度进行比较，包括对氧化应激的耐受性和在原代单核/巨噬细胞中的存活。3)确定突变体在体内致病过程中的贡献。感染性研究将在支持持续感染的SCID小鼠中进行，包括呼吸道内RSA439挑战的中度疾病过程。将建立竞争性感染模型，将突变体库中的感染能力和持久性与野生相比进行比较。使用荧光素酶表达的活体成像将方便地表征在动物中的复制和传播。对于选定的突变，将比较肺和脾的生长率和组织病理学后果。使用稳定的穿梭载体质粒对特定突变进行互补将证实，表型的改变是靶向转座子插入的结果，而不是第二次、未确定的突变。 公共卫生相关性：我们期望这些研究将导致一个优化的突变系统，用于未来对包括急性和慢性病毒型在内的全毒力分离株的研究。我们预测，大量的突变将表现为表型改变，并导致在获得性免疫缺陷但功能先天反应的小鼠身上识别对感染性、巨噬细胞存活以及生物体的定植和传播至关重要的基因。这些研究将有助于在这种生物的分子发病机制研究中建立一个根本性的范式转变。重要毒力决定因素的阐明将为疫苗设计、诊断抗原和治疗干预提供靶点。

DESCRIPTION (provided by applicant): Coxiella burnetii is a Gram-negative obligate intracellular bacterium that causes acute Q fever and chronic infections in humans. The two principal limitations for Q fever researchers have been 1) lack of genetic tools and 2) the obligate intracellular requirement for cultivation. Recent advances are beginning to resolve these restrictions: a) growth medium using microaerophilic conditions allowing replicate outside of host cells; b) novel techniques for efficient clonal isolation; c) functional Himar1-based transposon mutagenesis system to generate random mutations; and d) transformation with a stable shuttle vector plasmid. The objectives are to generate a mutant library of C. burnetii clones and evaluate phenotypic alteration in vitro and in vivo using C. burnetii, Nine Mile, phase II (RSA439, clone 4; reduced virulence, BL2 containment), as a model for future studies with fully virulent organisms by: 1) developing a transposon mutant clonal library for C. burnetii using a modified isolating clones by micromanipulation, Fluorescent Activated Cell Sorting (FACS) and propagated on agar plates. Each strategy will be optimized and contribute to an ongoing clonal library assembly. Mutants will be characterized genetically for location of insertion using whole genome amplification (WGA), Southern blot, PCR and rescue cloning. 2) Determine the contribution to the pathogenic process in vitro. Clones originating using both media and L929 fibroblast infection systems will be compared with wild type for growth rates in media and in several tissue culture model cells, including tolerance for oxidative stress and survival in primary monocytes/macrophages. 3) Determine the contribution to the pathogenic process for mutants in vivo. Infectivity studies will be performed in SCID mice, which support persistent infection including a moderate disease process with intratracheal RSA439 challenge. A competitive infection model will be established where a pool of mutants will be compared for ability to infect and persistent compared to wild. In vivo imaging using luciferase expression will conveniently characterize replication and dissemination in animals. Growth rates in and histopathologic consequences to lung and spleen will be compared for selected mutations. Complementation of specific mutation using the stable shuttle vector plasmid will confirm that alteration in phenotype is a result of the targeted transposon insertion and not a secondary, undefined mutation, . PUBLIC HEALTH RELEVANCE: It is our expectation that these studies will result in an optimized mutagenesis system for future studies with fully virulent isolates including both acute and chronic pathotypes. We predict that a large collection of mutations will manifest as altered phenotypes and result in identification of genes essential for infectivity, survival in macrophages and colonization and dissemination of organisms in a mouse with defective acquired immunity but functional innate responses. These studies will help establish a fundamental paradigm shift in the molecular pathogenesis studies with this organism. Elucidation of important virulence determinants will provide targets for vaccine design, diagnostic antigens, and therapeutic intervention.