Development of Site-Specific Mutagenesis in Coxiella burnetti

描述(申请人提供)：伯氏柯克斯体是一种革兰氏阴性的细胞内专性细菌，是人类急性和慢性Q(查询)热的病原体。伯氏梭菌毒力基因的分析由于无法产生和分离特定的突变而受到阻碍，到目前为止还依赖于异源宿主中基因表达的特征。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。该领域的最新进展，如胞外生长、明确的选择标记和穿梭载体，开始克服这些困难，并使现有的经典遗传方法能够适应和改进这种特定的微生物。该项目的目标是利用等位基因交换或乳酸杆菌移动第二组内含子LtrB的回巢机制(TargeTron技术，Sigma-Aldrich)在伯氏梭菌中开发有针对性的基因破坏。我们将通过以下具体目标来实现这一目标：(1)构建可调控伯氏梭菌基因表达的穿梭载体。在伯氏梭菌中，同源重组是由AddAB复合体介导的，而AddAB复合体的重组频率很低。为了克服这一障碍，我们将通过测试广泛使用的可诱导启动子的报告基因表达来建立受控的基因表达。这将支持引入异源重组系统和使用反选择标记来强制等位基因交换。(2)利用等位基因交换技术进行伯氏梭菌定点突变的研究。我们将在开发的柯克斯体特异性穿梭载体的基础上实现伯氏梭菌的等位基因交换。灭活基因将被运送到稳定复制的柯克斯体载体上，该载体具有可治愈的、对复制温度敏感的起始点。与经典的自杀载体相比，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系统进行伯氏梭菌定点突变的研究。我们将通过采用TargeTron技术，基于伯氏乳杆菌可重新编程的移动第二组内含子，实现有针对性的基因破坏。在所有三种方法中，我们都将针对基因，这些基因的失活导致可筛选的表型；rpsL的突变导致链霉素抗性和WAAF对脂多糖核心截断或作为免疫原性蛋白的COM1或SODC的失活。发展有针对性的基因破坏对伯氏梭菌来说将是新的，并对研究Q热致病过程的新方法做出了重大贡献。 与公共卫生相关：人类感染伯氏柯克斯体，一种潜在的生物恐怖主义因子，表现为急性流感样疾病或心脏或肝脏的慢性炎症。缺乏这种微生物的遗传工具，阻碍了对允许这种微生物感染并在人类体内存活的因素的识别。拟议的项目概述了开发基因功能表征的遗传工具的几项战略，并将有助于作为国家安全目标的疫苗的开发。

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.