Characterization of F. tularensis Virulence Genes

描述(申请人提供)：由于图拉氏方济氏菌易于传播，与肺炎图拉热症相关的低感染剂量和高死亡率，以及它在几个国家的生物武器计划中得到了深入的研究和开发，因此被认为是A类生物武器。关于最有可能的生物武器--图拉氏杆菌亚种的雾化形式的毒力，人们几乎一无所知。图拉尔人。本研究的重点是鉴定图拉氏丝孢霉亚种的所有必需基因和毒力基因。图拉尔通过基于基因组学的方法。这将极大地加深我们对图拉氏原虫致病机理的了解，并促进亚单位疫苗和减毒活疫苗的开发。首先，我们将利用一种名为GAMBIT的基因组学技术来诱变图拉曲霉亚种的每一个基因。然后将它们重新组合到染色体上。这将允许识别基本基因，这些基因是潜在的治疗靶点。存活的突变细菌将被组合在池中，通过气雾剂接种到小鼠体内，那些不能在宿主内生存的突变细菌将被识别出来。 通过基于微阵列的技术。这项技术将识别所有对图拉氏丝虫重要的基因 毒力通过气雾剂途径，其中一些可能是潜在的亚单位疫苗候选。其次是减毒的F·tularensis亚种。图拉尔病毒株将接受毒力标志的测试：巨噬细胞内生存和生长的能力，对抗菌化合物的抵抗力，以及入侵非吞噬细胞的能力。这将有助于鉴定和鉴定图拉氏丝孢子菌的关键毒力决定因素。最后，我们将对通过转录微阵列分析确定的毒力调节因子进行表征，包括转录激活因子mGIA，它应该能够阐明Francisella内的毒力调节因子，从而更好地理解致病的分子机制。我们的研究计划涉及多学科方法，并与组成该计划项目的其他项目进行高度整合。我们的联合专业知识将推动我们对图拉氏丝虫病的发病机制和免疫的了解，并使新的治疗和预防措施的开发成为可能。

DESCRIPTION (provided by applicant): Francisella tularensis is considered a Category A bioweapon due to the ease of transmission, the low infectious dose and high mortality associated with pneumonic tularemia, and the fact that it has been intensively studied and developed in bioweapons programs in several countries. Virtually nothing is known about virulence of aerosolized forms of the most likely bioweapon, F. tularensis subsp. tularensis. The focus of this research effort is to identify all the essential and virulence genes of F. tularensis subsp. tularensis through a genomics based approach. This will dramatically increase our understanding of F. tularensis pathogenesis, and facilitate the development of subunit and live-attenuated vaccines. First, we will utilize a genomics based technique termed GAMBIT to mutagenize every gene of F. tularensis subsp. tularensis, and then recombine them back onto the chromosome. This will allow for the identification of essential genes, which are potential therapeutic targets. The viable mutant bacteria will be combined in pools and inoculated via aerosol into mice, and those mutants that cannot survive within the host will be identified by a microarray-based technique. This technique will identify all genes important for F. tularensis virulence via the aerosol route, some of which may be potential subunit vaccine candidates. Next, the attenuated F. tularensis subsp. tularensis strains will be tested for markers of virulence: ability for intramacrophage survival and growth, resistance to antimicrobial compounds, and ability to invade nonphagocytic cells. This will allow for the identification and characterization of critical virulence determinants of F. tularensis. Finally, we will characterize the virulence regulatory factors identified by microarray analysis of transcription, including the transcriptional activator MgIA, which should illuminate virulence regulons within Francisella, allowing a better understanding of the molecular mechanisms of pathogenesis. Our research plan involves a multidisciplinary approach and high levels of integration with the other projects that compose this program project. Our combined expertise will propel our knowledge of F. tularensis pathogenesis and immunity and allow for the development of novel therapeutic and preventive measures.