Regulation of Gene Expression in Francisella

图拉氏方济各氏菌会在动物和人类中引起严重感染。这种革兰氏阴性兼性 胞内细菌易于体外培养，高毒力菌株的感染量仅为10 到50个活细胞。图拉氏丝虫可以通过包括破皮在内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。 图拉拉人感染。这些特性，以及图拉氏丝虫过去被武器化的事实，增加了 开发疫苗和确定治疗目标的紧迫性。长期的 这项建议的目标是确定在巨噬细胞中对图拉氏丝虫细胞内复制至关重要的基因。 细胞内复制所需的基因在模型哺乳动物宿主和在 对人类的感染。为了实现这一目标，我们报道了一系列将 允许鉴定和表征有助于细胞内存活的弗朗西斯菌基因 活体表达接近了。巨噬细胞诱导的基因将被特定的等位基因敲除 LVS和一株A型强毒株的交换策略。由此产生的菌株将被表征为 巨噬细胞和小鼠感染模型中的毒力相关表型。描述了新的 已确定的体内表达基因和计划中的PurC和AroA敲除有望提供 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屏幕预计将 确定控制细胞内复制的结构基因和调控基因。了解监管机构 允许感染和繁殖的途径对于破译由 细菌。将利用微阵列分析来研究调节级联反应中的基因联系。 总体而言，我们建议结合表达技术和全局分析来鉴定毒力 与细胞内复制相关的决定因素，在特征不佳的选择因子中，F。

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.