Molecular elucidation of the Francisella tularensis virulence mechanism

土拉弗朗西斯菌毒力机制的分子阐明

• 批准号: 10408864
• 负责人: RICHARD GERALD BRENNAN
• 金额: $ 79.71万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-21 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10408864
• 关键词: Aerosols; Affinity; Automobile Driving; Bacteria; Binding; Biochemical; Biological Assay; Blood capillaries; C-terminal; Categories; Cells; Complex; Cryoelectron Microscopy; Crystallization; DNA; DNA Binding; DNA Structure; DNA-Binding Proteins; Dangerousness; Data; Development; Disease Outbreaks; Dissection; Drug Design; Drug Targeting; Elements; Fluorescence Polarization; Francisella; Francisella tularensis; Gene Expression; Genes; Goals; Government; Growth; Guanosine Tetraphosphate; Helix-Turn-Helix Motifs; Heterodimerization; Holoenzymes; Ligands; Mediating; Medical center; Molecular; Morbidity - disease rate; Pathogenesis; Pathogenicity; Pathogenicity Island; Peptides; Polymerase; Proteins; Radial; Regulation; Regulator Genes; Regulon; Research; Resolution; Signal Transduction; Site; Starvation; Structure; Tail; Testing; Therapeutic; Transcriptional Activation; Transcriptional Regulation; Tularemia; Virulence; Winged Helix; aerosolized; base; bioweapon; improved; in vivo; inhibitor; macrophage; mortality; new therapeutic target; novel; novel therapeutics; pathogenic bacteria; prevent; promoter; protein expression; rational design; recruit; transcriptome sequencing

Francisella tularensis (Ft), the causative agent of tularemia, is one of the most infectious bacterial pathogens known. Due to its high infectivity and ease of aerosolization, it has been classified as Category A bioweapon by the US government. The morbidity and mortality of tularemia are substantial, and given its extreme infectivity, a significant outbreak of tularemia would readily overwhelm the capabilities of even the largest US medical centers. Ft virulence requires genes expressed from the chromosomally encoded Ft pathogenicity island (FPI). The expression of these genes are activated by a combination of Ft regulators: the stringent starvation protein A (SspA), the macrophage growth locus protein A (MglA) and the pathogenicity island gene regulator (PigR), which are expressed during Ft infection. MglA and PigR are unique to Ft whereas SspA proteins are found in multiple bacteria. The Ft SspA, however, is unusual as it does not homodimerize but rather heterodimerizes with MglA. PigR is a DNA-binding protein with a predicted winged-helix-turn-helix DNA-binding motif. Intriguingly, the “alarmone”, guanosine tetraphosphate (ppGpp), is also necessary for Ft virulence. Recently, we showed that this alarmone binds directly to the MglA-SspA complex. We further showed that ppGpp binding to MglA-SspA mediates high-affinity binding of PigR to this heterodimer. Strikingly, our data also revealed that MglA-SspA interacts constitutively with the Ft RNAPs70 holoenzyme suggesting it represents a virulence specialized RNAP. Given the extreme virulence and potential use of Ft as a bioweapon, there is an urgent need to decipher the molecular mechanisms driving its virulence. The overarching goal of this proposal is the molecular dissection of these mechanisms. Our central hypothesis is that Ft employs a conceptually novel form of pathogenesis requiring a virulence-specialized RNAP containing MglA-SspA. We shall test our central hypothesis and delineate the molecular mechanisms controlling the activation of Ft virulence genes through the completion of three Specific Aims: Specific Aim 1: To fully characterize MglA-SspA interaction with Ft RNAP. Structural, biochemical and cellular studies will dissect the mechanism of virulence regulation by MglA- SspA. Specific Aim 2: To carry out structure and function analyses of Ft RNAP(MglA-SspA)-ppGpp-PigR complexes. The mechanism behind PigR-mediated activation of the FPI will be analyzed structurally, biochemically and in vivo. Specific Aim 3: To determine a high resolution (MglA-SspA)-ppGpp-PigR crystal structure, identify inhibitors of ppGpp binding to MglA-SspA and obtain structures of MglA-SspA inhibitor complexes. The successful completion of these Aims will reveal new paradigms in transcription regulation and enable the rational design of novel anti Francisella-virulence therapeutics.

francisella tularensis（ft）是tularemia的致病药物，是最感染性细菌病原体之一 已知。由于其高感染和易于气化，因此已将其归类为生物武器类别 美国政府。 t骨的发病率和死亡率是实质的，鉴于其极端的感染， 大量爆发tularemia很容易使美国医学最大的医学的能力不堪重负 中心。 FT病毒需要从染色体编码的FT致病岛（FPI）表达的基因。 这些基因的表达是通过FT调节剂的组合激活的：严格的饥饿蛋白 A（SSPA），巨噬细胞生长基因座蛋白A（MGLA）和致病性岛基因调节剂（PIGR）， 在英尺感染期间表达。 MGLA和PIGR是FT独有的，而SSPA蛋白在 多种细菌。但是，FT SSPA是不寻常的 与mgla。 PIGR是一种DNA结合蛋白，具有预测的翼螺旋 - 螺旋螺旋DNA结合基序。 有趣的是，“警报器”，四磷酸鸟嘌呤四磷酸（PPGPP）也是FT病毒所必需的。最近， 我们表明该警报器直接与MGLA-SSPA复合物结合。我们进一步表明PPGPP结合 为了MGLA-SPA介导PIGR与该异二聚体的高亲和力结合。令人惊讶的是，我们的数据还显示 MGLA-SPA与FT RNAPS70全酶组成构成相互作用，表明它代表了病毒 专门的RNAP。考虑到FT作为生物武器的极端病毒和潜在用途，有一个紧急的 需要破译驱动其病毒的分子机制。该提议的总体目标是 这些机制的分子解剖。我们的中心假设是，英尺雇员在概念上新颖的形式 需要病毒特异性RNAP的发病机理。我们将测试我们的中央 假设并描述控制FT病毒基因激活的分子机制 完成三个特定目的的完成：特定目标1：完全表征与ft的MGLA-SSPA相互作用 rnap。结构，生化和细胞研究将剖析MGLA-的病毒调节机制 SSPA。特定目标2：进行FT RNAP（MGLA-SSPA）-PPGPP-PIGR的结构和功能分析 复合物。 PIGR介导的FPI激活背后的机制将在结构上分析， 生化和体内。特定目标3：确定高分辨率（MGLA-SSPA）-PPGPP-PIGR晶体 结构，鉴定PPGPP与MGLA-SSPA结合的抑制剂，并获得MGLA-SSPA抑制剂的结构 复合物。这些目标的成功完成将揭示转录法规中的新范式， 启用新型抗Francisella病毒疗法的合理设计。