Regulation of Gene Expression in Francisella

土拉热弗朗西丝氏菌在动物和人类中引起严重感染。这种革兰氏阴性兼性 胞内细菌易于在体外培养，强毒株的感染剂量仅为10 50个活细胞F.土拉热可以通过包括破损皮肤的几个入口进入宿主， 眼、喉和肺的粘膜上皮。目前还没有获得许可的疫苗来预防F。 土拉菌感染这些性质以及F.土拉热在过去被武器化， 开发疫苗和确定治疗靶点的紧迫性。长期 该计划的目的是鉴定对F.巨噬细胞中的土拉菌。 细胞内复制所需的基因可能在模型哺乳动物宿主中和在哺乳动物宿主中是重要的。 人类感染。为了实现这一目标，我们报告了一系列质粒载体的构建， 允许鉴定和表征弗朗西斯菌基因有助于细胞内存活， 体内表达方法。巨噬细胞诱导的基因将被等位基因特异性敲除， LVS交换策略和毒性A型菌株。将对所得菌株进行以下表征： 巨噬细胞和小鼠感染模型中的毒力相关表型。新特性 鉴定的体内表达基因和计划的purC和aroA敲除预期提供了一种新的方法， 为未来的活疫苗试验选择合适的菌株。我们的巨噬细胞报告基因筛选有望 鉴定控制细胞内复制的结构和调节基因。了解监管 允许感染和繁殖的途径对于破译由细菌感知的环境线索至关重要。 细菌。将利用微阵列分析研究调控级联中的基因连锁。 总之，我们建议将联合收割机表达技术和全局分析相结合来鉴定毒力 决定因素与细胞内复制的特点不佳的选择剂，F。土拉热。

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.