Regulation of Gene Expression in Francisella

描述（由申请人提供）：土拉菌在动物和人类中引起严重感染。这种革兰氏阴性兼性胞内细菌很容易在体外培养，高毒力菌株的感染剂量仅为10至50个活细胞。土拉菌可通过破损的皮肤、眼睛、喉咙和肺部的粘膜上皮等途径进入宿主体内。目前还没有获得许可的预防土拉菌感染的疫苗。这些特性，以及过去土拉菌被武器化的事实，增加了开发疫苗和确定治疗靶点的紧迫性。本研究的长期目标是确定巨噬细胞中土拉菌胞内复制的重要基因。细胞内复制所需的基因在模式哺乳动物宿主和人类感染过程中可能很重要。为了实现这一目标，我们报道了一系列质粒载体的构建，这些质粒载体将允许使用体内表达方法鉴定和表征有助于细胞内存活的Francisella基因。巨噬细胞诱导的基因将在LVS和毒力强的a型菌株中通过等位基因交换策略被特异性敲除。由此产生的菌株将在巨噬细胞和小鼠感染模型中以毒力相关表型为特征。新发现的体内表达基因的特征和计划的purC和aroA敲除预计将为未来的活疫苗试验提供合适的菌株选择。我们的巨噬细胞报告筛选有望识别控制细胞内复制的结构和调控基因。了解允许感染和繁殖的调控途径对于破译细菌感知的环境线索至关重要。基因的连锁调节级联将利用微阵列分析进行调查。总之，我们建议结合表达技术和全局分析来确定与特征不佳的选择剂土拉菌胞内复制相关的毒力决定因素。

DESCRIPTION (provided by applicant): Francisella tularensis causes severe infections in animals and humans. This Gram-negative facultative intracellular bacterium is easily cultivated in vitro and the infectious dose of highly virulent strains is only 10 to 50 viable cells. F. tularensis can gain entry to the host through several portals that include broken skin, the mucosal epithelium of the eye, throat, and lungs. There is no licensed vaccine to protect against F. tularensis infections. These properties, and the fact that F. tularensis was weaponized in the past, increase the urgency towards the development of vaccines and the identification of therapeutic targets. The long-term goal of this proposal is to identify genes important for intracellular replication of F. tularensis in macrophage. Genes required for intracellular replication are likely important in model mammalian hosts and during infections in humans. Towards this goal we report the construction of a series of plasmid vectors that will allow the identification and characterization of Francisella genes contributing to intracellular survival using in vivo expression approaches. Macrophage-induced genes will be specifically knocked-out with an allelic exchange strategy in LVS and a virulent type A strain. The resulting strains will be characterized for virulence-associated phenotypes in macrophage and mouse models of infection. Characterization of newly identified in vivo expressed genes and planned purC and aroA knock-outs is anticipated to provide a selection of suitable strains for future live vaccine trials. Our macrophage reporter screen is expected to identify both structural and regulatory genes governing intracellular replication. Understanding the regulatory pathways allowing infection and multiplication is critical to deciphering the environmental cues sensed by the bacterium. The linkage of genes in regulatory cascades will be investigated utilizing microarray analyses. Overall, we propose to combine expression technology and global analyses to identify virulence determinants associated with intracellular replication in the poorly characterized select agent, F. tularensis.