Development of Site-Specific Mutagenesis in Coxiella burnetti

描述（由申请人提供）：伯纳克希菌是一种革兰氏阴性专性细胞内细菌，是人类急性和慢性Q （query）热的病原。由于无法产生和分离特异性突变，迄今为止，对伯氏疏螺旋体毒力基因的分析一直受到阻碍，并且依赖于外源宿主中基因表达的表征。10多年前首次报道了稳定维持的外源质粒DNA的引入，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。最近在细胞外生长、定义选择标记和穿梭载体等领域的进展开始克服这些困难，并允许对现有的经典遗传方法进行适应和改进，以适应这种特定的微生物。该项目的目的是利用乳酸菌II族可移动内含子LtrB的等位基因交换或后归巢机制（TargeTron技术，Sigma-Aldrich），在伯氏杆菌中开发靶向基因破坏。我们将通过追求以下具体目标来实现这一目标：(1)开发一种用于控制伯氏梭菌基因表达的穿梭载体。在burnetii中，同源重组是由AddAB复合物介导的，其重组频率较低。为了克服这一障碍，我们将通过检测广泛使用的诱导启动子的报告基因表达来建立受控基因表达。这将为引入异源重组系统和利用反选择标记强制等位基因交换提供支持。(2)利用等位基因交换技术进行burnetii位点特异性诱变。我们将基于开发的coxiella特异性穿梭载体实现伯氏梭菌的等位基因交换。灭活的基因将被递送到具有可治愈的、复制温度敏感源的稳定复制的科希氏菌载体上。与传统的自杀载体相比，这种方法在强制整合事件之前提供了更多的底物DNA，并可能导致更高的成功率。(3)利用TargeTron系统开发burnetii位点特异性诱变技术。我们将采用TargeTron技术，基于L. lactis对burnetii的可重新编程的移动II族内含子，实现靶向基因破坏。在这三种方法中，我们将专门针对基因，其失活导致可筛选表型；rpsL突变导致链霉素耐药和waaF对LPS核心截断或com1或sodC作为替代免疫原性蛋白失活。靶向基因破坏的发展将是伯纳氏杆菌的新方法，并为研究Q热的致病过程提供了新的途径。

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.