Iron uptake mechanisms of francisella tularensis

土拉弗朗西斯菌的铁吸收机制

• 批准号: 7446767
• 负责人: GIRIJA RAMAKRISHNAN
• 金额: $ 33.44万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7446767
• 关键词: Anabolism; Animal Model; Antibodies; Attenuated; Bacteria; Binding; Biochemical; Biological Assay; Biological Models; Bioterrorism; Carboxy-Lyases; Cell Fractionation; Cells; Complex; Condition; Culture Media; Cytochromes; Cytoplasm; Dependence; Development; Disease; Disease Outbreaks; Environment; Enzymes; Escherichia coli; Ferritin; Francisella tularensis; Future; Generations; Genes; Genome; Goals; Gram-Negative Bacteria; Growth; Hemin; Hemoglobin; Homologous Gene; Homologous Protein; Immunoelectron Microscopy; Immunologic Techniques; Indirect Immunofluorescence; Indium; Infection; Iron; Iron Chelating Agents; Ligase; Mediating; Membrane; Membrane Proteins; Mutagenesis; Mutation; Nature; Nutrient; Operon; Organism; Pathogenesis; Pathway interactions; Phagosomes; Phenotype; Plasmids; Production; Protein Family; Proteins; RNA; Recombinants; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Route; Siderophores; Source; Staging; Structural Protein; Subunit Vaccines; System; Testing; Therapeutic; Transferrin; Tularemia; Vaccines; Virulence; Western Blotting; base; deletion analysis; genetic analysis; intraperitoneal; macrophage; microbial; microorganism; mouse model; mutant; novel; pathogen; pathogenic bacteria; polyclonal antibody; programs; protein expression; reconstitution; siderophore receptors; small molecule; uptake

DESCRIPTION (provided by applicant): Francisella tularensis is a gram-negative bacterium and the causative agent of tularemia, a potentially deadly disease. It is a threat as an agent of bioterrorism because of the highly infectious nature of the airborne organism. There is not currently an approved vaccine to protect against this agent. In the mammalian host, the bacterium is an intracellular pathogen of macrophages and other cells; they reside within membrane-bound phagosomes early in infection and subsequently escape into the cytoplasm where they replicate. Iron acquisition systems in the iron-scarce host environment are critical virulence determinants in many pathogens. Although iron is important for proliferation of F. tularensis within macrophages, iron acquisition mechanisms in this organism are uncharacterized. This proposal is based on the hypothesis that iron acquisition is a key element in pathogenesis in the mammalian host. Many microorganisms secrete small molecules called siderophores to chelate iron from a variety of sources. We have shown that the F. tularensis bacteria secrete a siderophore whose production is dependent on the fsIA gene. Iron-siderophore uptake by gram-negative bacteria is universally dependent on the TonB-ExbB-ExbD complex, but the F. tularensis genome does not encode homologs of these proteins. We have determined that the fslE gene is necessary for iron acquisition under conditions of iron limitation and from host sources. The goal of this proposal is to investigate the roles of the fslABCDEF genes in iron uptake in F. tularensis. Specific aim 1 of this project is the phenotypic analysis of deletion mutants in each of the fsl genes. We will test for siderophore expression and iron uptake, and we will also assess the virulence of the mutants in a mouse model of infection. Specific aim 2 is the characterization of the Fsl gene products. We will determine the subcellular localization of each of the gen products using biochemical and immunological techniques. We will characterize the activity of the putative enzymes FsIA and FslC. We will test the ability to reconstitute the iron acquisition system involving FslE and/or FsIF in Escherichia coli as a model system to study a novel paradigm for iron uptake. Understanding the mechanisms to acquire iron will enable us to harness them in therapeutic applications and in development of defined subunit vaccine and vaccine strains so that we may be better prepared to deal with a potential tularemic outbreak.

描述（由申请人提供）：土拉弗朗西斯菌是一种革兰氏阴性细菌，是土拉菌病（一种潜在致命疾病）的病原体。由于空气中的生物体具有高度传染性，因此它是一种生物恐怖主义的威胁。目前还没有批准的疫苗可以预防这种物质。在哺乳动物宿主中，细菌是巨噬细胞和其他细胞的细胞内病原体；它们在感染早期驻留在膜结合的吞噬体内，随后逃入细胞质并在那里进行复制。缺铁宿主环境中的铁获取系统是许多病原体的关键毒力决定因素。尽管铁对于土拉弗朗西斯巨噬细胞内的增殖很重要，但该生物体中铁的获取机制尚不清楚。该提议基于这样的假设：铁的获取是哺乳动物宿主发病机制的关键因素。许多微生物分泌称为铁载体的小分子来螯合各种来源的铁。我们已经证明土拉弗朗西斯细菌分泌铁载体，其产生依赖于 fsIA 基因。革兰氏阴性细菌对铁铁载体的摄取普遍依赖于 TonB-ExbB-ExbD 复合物，但土拉弗朗西斯菌基因组不编码这些蛋白质的同源物。我们已经确定，fslE 基因对于铁限制条件下和从宿主来源获取铁是必需的。本提案的目的是研究 fslABCDEF 基因在土拉弗拉菌铁吸收中的作用。该项目的具体目标 1 是对每个 fsl 基因中的缺失突变体进行表型分析。我们将测试铁载体表达和铁吸收，我们还将评估突变体在小鼠感染模型中的毒力。具体目标 2 是 Fsl 基因产物的表征。我们将使用生化和免疫学技术确定每种基因产物的亚细胞定位。我们将表征推定酶 FsIA 和 FslC 的活性。我们将测试在大肠杆菌中重建涉及 FslE 和/或 FsIF 的铁获取系统的能力，作为模型系统来研究铁吸收的新范例。了解获取铁的机制将使我们能够在治疗应用中利用它们以及开发特定的亚单位疫苗和疫苗株，以便我们更好地准备应对潜在的土拉菌病爆发。