Characterization of F. tularensis Virulence Genes

描述（由申请人提供）：土拉菌被认为是a类生物武器，因为它易于传播，与肺炎土拉菌相关的低感染剂量和高死亡率，而且它已经在几个国家的生物武器计划中得到了深入研究和开发。实际上，人们对最可能的生物武器土拉菌亚种的雾化形式的毒力一无所知。土拉杆菌内。本研究的重点是鉴定土拉菌亚种的所有必需和毒力基因。通过基于基因组学的方法。这将大大增加我们对土拉菌病发病机制的了解，并促进亚单位和减毒活疫苗的开发。首先，我们将利用基于基因组学的技术称为GAMBIT来诱变土拉菌亚种的每个基因。土拉菌，然后将它们重新组合到染色体上。这将允许识别基本基因，这是潜在的治疗靶点。有活力的突变细菌将在池中组合，并通过气溶胶接种到老鼠身上，那些不能在宿主体内存活的突变细菌将被识别出来

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.