Regulation of Gene Expression in Francisella

弗朗西斯菌基因表达的调控

• 批准号: 7031430
• 负责人: Dara W. Frank
• 金额: $ 37.88万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-15 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031430
• 关键词: clinical research; gene expression; genes; human; infection; intracellular; macrophage; model; virulence

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.

描述（由申请人提供）：土拉弗朗西斯菌引起动物和人类的严重感染。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。土拉弗朗西斯菌可以通过多个入口进入宿主，包括破损的皮肤、眼睛的粘膜上皮、喉咙和肺部。目前还没有获得许可的疫苗可以预防土拉弗拉菌感染。这些特性，以及土拉杆菌过去被武器化的事实，增加了开发疫苗和识别治疗靶点的紧迫性。该提案的长期目标是鉴定对巨噬细胞中土拉弗朗西斯菌细胞内复制重要的基因。细胞内复制所需的基因可能在模型哺乳动物宿主和人类感染过程中很重要。为了实现这一目标，我们报告了一系列质粒载体的构建，这些载体将允许使用体内表达方法来鉴定和表征有助于细胞内存活的弗朗西斯菌基因。巨噬细胞诱导的基因将通过 LVS 和强毒力 A 型菌株中的等位基因交换策略被特异性敲除。所得菌株将在巨噬细胞和小鼠感染模型中表征毒力相关表型。新鉴定的体内表达基因和计划的 purC 和 aroA 敲除的表征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基因筛选有望识别控制细胞内复制的结构基因和调节基因。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。将利用微阵列分析来研究调控级联中基因的联系。总的来说，我们建议将表达技术和全局分析相结合，以确定与特征较差的选择剂土拉弗朗西斯细胞内复制相关的毒力决定因素。