Regulation of Gene Expression in Francisella

弗朗西斯菌基因表达的调控

• 批准号: 7560339
• 负责人: Dara W. Frank
• 金额: $ 36.08万
• 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/7560339
• 关键词: Adherence; Animal Model; Animals; Attenuated Vaccines; Bacteria; Biological; Candidate Disease Gene; Categories; Cells; Centers for Disease Control and Prevention (U.S.); Clinical; Cues; DNA; Dose; Epithelium; Escherichia coli; Eye; Francisella; Francisella tularensis; Future; Gene Expression Regulation; Genes; Genetic; Goals; Growth; Human; In Vitro; Infection; Knock-out; Licensing; Lung; Mediating; Modeling; Nature; Organism; Oryctolagus cuniculus; Parasites; Pathway interactions; Pharyngeal structure; Phenotype; Plasmid Cloning Vector; Property; Proteins; Regulator Genes; Regulatory Pathway; Reporter; Reporting; Rodent; Series; Severity of illness; Skin; Symptoms; System; Techniques; Technology; Tularemia; Vaccines; Virulence; Virulent; attenuation; density; in vivo; interest; macrophage; mouse model; mutant; pathogen; programs; response; therapeutic target; tissue culture; tool; trafficking; uptake; vaccine development; weapons

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.

土拉弗朗西斯菌会引起动物和人类的严重感染。这种革兰氏阴性兼性 胞内菌易于体外培养，高毒力菌株感染剂量仅为10 至 50 个活细胞。 F. tularensis 可以通过多个入口进入宿主，这些入口包括破损的皮肤、 眼睛、喉咙和肺的粘膜上皮。没有获得许可的疫苗可以预防 F. 土拉热丝虫感染。这些特性以及 F. tularensis 过去被武器化的事实，增加了 开发疫苗和确定治疗靶点的紧迫性。长期来看 该提案的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在模型哺乳动物宿主和复制过程中可能很重要 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，这些载体将 允许鉴定和表征有助于细胞内存活的弗朗西斯菌基因，用于 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 和强毒力 A 型菌株的交换策略。所得菌株将被表征为 巨噬细胞和小鼠感染模型中毒力相关的表型。新的表征 鉴定的体内表达基因和计划的 purC 和 aroA 敲除预计将提供 选择合适的菌株用于未来的活疫苗试验。我们的巨噬细胞报告屏幕预计 识别控制细胞内复制的结构基因和调节基因。了解监管 允许感染和繁殖的途径对于破译病毒感知到的环境线索至关重要 细菌。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，我们建议结合表达技术和全局分析来识别毒力 与特征不明的选择因子土拉弗朗西斯 (F. tularensis) 细胞内复制相关的决定因素。