Development of Site-Specific Mutagenesis in Coxiella burnetti

描述（由申请方提供）：贝氏柯克斯体是一种革兰氏阴性专性细胞内细菌，是人类急性和慢性Q（查询）热的病原体。分析了C.贝氏体毒力基因的研究由于不能产生和分离特异性突变而受到阻碍，并且迄今为止依赖于异源宿主中基因表达的表征。稳定维持的外源质粒DNA的引入在10多年前首次报道，但随后的报道和未报道的利用这种穿梭载体的尝试令人失望。该领域的最新进展，如细胞外生长，确定的选择标记和穿梭载体开始克服这些困难，并允许适应和完善现有的经典遗传方法，这种特定的微生物。本项目的目标是在C.使用等位基因交换或乳酸乳杆菌的移动的II组内含子LtrB的回巢机制（Targetron technology，Sigma-Aldrich），本论文的主要研究内容如下：（1）构建穿梭载体，在大肠杆菌中进行基因表达调控;伯内特氏菌In C.贝氏体同源重组由AddAB复合物介导，其与低重组频率相关。为了克服这一障碍，我们将通过检测报告基因在C.伯内特氏菌这将支持异源重组系统在C. Burnetii和使用反向选择标记来强制等位基因交换。(2)定点突变在C. burnetii使用等位基因交换。我们将在C中实现等位基因交换。贝氏柯克斯体的基础上开发的柯克斯体特异性穿梭载体。失活基因将在具有可治愈的复制温度敏感性起点的稳定复制型柯克斯体载体上递送。当与经典自杀载体相比时，该方法在强制整合事件之前提供了更高量的底物DNA，并且可能导致更高的成功率。(3)定点突变在C. Burnetii使用Targetron系统。我们将通过采用Targetron技术实现靶向基因破坏，该技术基于L. lactis为C.伯内特氏菌在所有三种方法中，我们将特异性靶向基因，其失活导致可筛选的表型; rpsL的突变导致链霉素抗性和waaF对LPS核心截短或com 1或sodC作为免疫原性蛋白的失活作为替代。靶向基因破坏的发展将是新的C。burnetii和一个重大贡献的新方法来调查的致病过程Q热。 公共卫生相关性：人类感染贝氏柯克斯体是一种潜在的生物恐怖主义制剂，表现为急性流感样疾病或心脏或肝脏的慢性炎症。由于缺乏这种微生物的遗传工具，阻碍了对允许该生物体感染并在人类中持续存在的因素的鉴定。拟议的项目概述了开发基因工具以确定基因功能的几项战略，并将帮助开发作为国家安全目标的疫苗。

DESCRIPTION (provided by applicant): Coxiella burnetii, a Gram-negative obligate intracellular bacterium, is the etiological agent of acute and chronic Q (query) fever in humans. Analysis of C. burnetii virulence genes has been hampered by the inability to generate and isolate specific mutations and relies to date on the characterization of gene expression in heterologous hosts. Introduction of stably maintained exogenous plasmid DNA was first reported over 10 years ago, but subsequent reports and unreported attempts to utilize this shuttle vector were disappointing. Recent advances in the field such as, extracellular growth, defined selection markers and a shuttle vector start to overcome these difficulties and allow adaptation and refinement of existent classical genetic methods to this specific microorganism. The objective of this project is to develop targeted gene disruption in C. burnetii using allelic exchange or the retrohoming mechanism of the mobile group II intron LtrB of Lactobacillus lactis (TargeTron technology, Sigma-Aldrich). We will accomplish the objective by pursuing the following specific aims: (1) Development of a shuttle vector for controlled gene expression in C. burnetii. In C. burnetii homologous recombination is mediated by the AddAB complex, which is associated with a low recombination frequency. To overcome this hurdle we will establish controlled gene expression by testing reporter gene expression from widely used inducible promoter in C. burnetii. This will support the introduction of heterologous recombination systems in C. burnetii and usage of counter selection markers to force allelic exchange. (2) Development of site-specific mutagenesis in C. burnetii using allelic exchange. We will achieve allelic exchange in C. burnetii based on the developed Coxiella-specific shuttle vector. Inactivated genes will be delivered on the stable replicating Coxiella vector with a curable, for replication temperature-sensitive origin. This approach provides a higher amount of substrate DNA, when compared to classical suicide vectors, prior to the forced integration event and might lead to a higher success rate. (3) Development of site-specific mutagenesis in C. burnetii using the TargeTron system. We will achieve targeted gene disruption by adapting the TargeTron Technology, based on the re-programmable mobile group II intron of L. lactis for C. burnetii. In all three approaches we will specifically target genes, which inactivation results in a screenable phenotype; Mutation of rpsL results in streptomycin resistance and inactivation of waaF to LPS core truncation or com1 or sodC as immunogenic proteins as alternatives. Development of targeted gene disruption would be novel for C. burnetii and a major contribution to new approaches to investigate the pathogenic process of Q fever. PUBLIC HEALTH RELEVANCE: Infections of humans with Coxiella burnetii, a potential bioterrorism agent, manifest as acute, flu-like illness or as chronic inflammation of the heart or liver. The lack of genetic tools for this microorganism has hampered the identification of factors which allow the organism to infect and persist in humans. The proposed project outlined several strategies to develop genetic tools for characterization of gene functions and will aid the development of a vaccine as national security goal.