Structural Elucidation of the Novel RNA Polymerase Underlying Francisella Tularensis Virulence

土拉弗朗西斯菌毒力背后的新型 RNA 聚合酶的结构解析

• 批准号: 9977601
• 负责人: RICHARD GERALD BRENNAN
• 金额: $ 23.42万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977601
• 关键词: Affinity; Automobile Driving; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Categories; Cells; ChIP-seq; Complex; Cryoelectron Microscopy; DNA-Binding Proteins; DNA-Directed RNA Polymerase; Dangerousness; Development; Disease Outbreaks; Dissection; Docking; Fluorescence Polarization; Francisella; Francisella tularensis; Gene Activation; Genes; Genetic Transcription; Goals; Government; Growth; Guanosine Tetraphosphate; Helix-Turn-Helix Motifs; Heterodimerization; Holoenzymes; Human; Infection; Infectious Agent; Knowledge; Laboratories; Maps; Mediating; Medical center; Molecular; Morbidity - disease rate; Pathogenicity; Pathogenicity Island; Polymerase; Proteins; RNA Polymerase I; RNA Polymerase II; Regulator Genes; Research; Resolution; Signal Transduction; Starvation; State Government; Stress; Structure; System; Tail; Testing; Therapeutic; Transcriptional Activation; Transcriptional Regulation; Tularemia; United States; Virulence; Virulence Factors; Winged Helix; aerosolized; bioweapon; cell type; design; experimental study; inhibitor/antagonist; insight; macrophage; man; mortality; new therapeutic target; novel; novel therapeutics; pathogenic bacteria; programs; promoter; recruit; structural biology

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known. This bacterium can be readily aerosolized and utilized as a bioweapon. The morbidity and mortality of tularemia are significant and, given the infectious capability of Francisella, a major outbreak would readily overwhelm the ability of even the largest U.S. medical centers. Consequently, Francisella is classified as a category A bioweapon by the US government. Genes encoded on the Francisella pathogenicity island (FPI), are responsible for the virulence of this bacterium. The stringent starvation protein A (SspA), the macrophage growth locus protein A (MglA) and the pathogenicity island gene regulator (PigR) mediate activation of these genes and are therefore essential for the virulence of Francisella species that infect humans. MglA and PigR are unique to Francisella whereas SspA proteins are found in multiple bacteria. The Francisella SspA, however, is unusual in that it does not homodimerize but rather functions as a heterodimer with MglA. PigR is a putative DNA binding protein with a predicted winged-helix-turn-helix motif. How SspA-MglA and PigR mediate FPI activation is unknown, as is the underlying molecular mechanism that these proteins use to sense infection. The overarching goal of this proposal is the molecular dissection of these mechanisms through the study of these virulence factors in the human pathogenic Francisella tularensis tularensis and holartica subspecies. Early studies implicated the “alarmone”, guanosine-tetraphosphate (ppGpp), as key for Francisella virulence. We recently showed that ppGpp binds directly to MglA-SspA and unveiled the molecular details of this interaction by solving the MglA-SspA-ppGpp complex structure. Further, we showed that ppGpp binding to MglA-SspA mediates high affinity binding of PigR to this heterodimer. In this revised proposal, we shall leverage our recent discoveries to dissect all components of the Francisella virulence regulatory system, including critically, the Francisella RNA polymerase (RNAP). Our central hypothesis is that F. tularensis employs a conceptually novel form of virulence activation involving a unique RNAP that contains the virulence activating complex MglA-SspA as a core constituent. This is supported by ChIP-seq studies and RNAP purifications from Francisella cells. We shall test our central hypothesis and complete the proposed objectives through two Specific Aims. Specific Aim 1: Elucidate the high resolution structure of (MglA-SspA)-ppGpp-PigR and identify inhibitors of ppGpp binding to MglA-SspA. Specific Aim 2: Determine the structure of Francisella RNAP complexes by cryo-EM. The successful completion of these studies will reveal a new paradigm in transcription regulation and enable the rational design of novel anti-Francisella-virulence therapeutics.

francisella tularensis是tularemia的病因，是最感染性细菌病原体之一 已知。该细菌可以很容易被雾化并用作生物武器。发病率和死亡率 Tularemia很重要，鉴于Francisella的感染能力，很容易爆发 即使是最大的美国医疗中心的能力，也压倒了能力。因此，弗朗西斯拉被归类为 美国政府的生物武器类别。在Francisella致病岛（FPI）上编码的基因， 负责该细菌的病毒。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）介导了这些激活 基因，因此对于感染了人类的弗朗西斯氏菌的病毒至关重要。 MGLA和PIGR 弗朗西斯菌是独有的，而SSPA蛋白在多种细菌中发现。 Francisella SSPA， 但是，这是不寻常的，因为它不会同构二聚体，而是作为MGLA的异二聚体起作用。 Pigr是一个 推定的DNA结合蛋白具有预测的翼螺旋 - 螺旋螺旋序。 SSPA-MGLA和PIGR如何介导 FPI激活尚不清楚，这些蛋白质用来感知的基本分子机制也是 感染。该提案的总体目标是通过这些机制的分子解剖 研究这些病毒因子在人类致病性francisella tularensis和holartica中的研究 亚种。早期研究牵涉到“警报器”，鸟苷 - 四磷酸盐（PPGPP），作为弗朗西斯拉的关键 病毒。我们最近表明，PPGPP直接与MGLA-SSPA结合，并揭示了分子细节 通过求解MGLA-SSPA-PPGPP复合结构的相互作用。此外，我们表明PPGPP与 MGLA-SPA介导了PIGR与该异二聚体的高亲和力结合。在这项修订的建议中，我们将 利用我们最近的发现来剖析弗朗西斯氏病毒调节系统的所有组成部分， 包括批判性的Francisella RNA聚合酶（RNAP）。我们的中心假设是F. tularensis 员工一种概念性小说的病毒激活形式涉及一个独特的RNAP，其中包含病毒 激活复杂的MGLA-SSPA作为核心。这得到了Chip-Seq研究和RNAP的支持 Francisella细胞的纯化。我们将检验我们的中心假设并完成提议的目标 通过两个具体的目标。特定目标1：阐明（MGLA-SPA）-PPGPP-PIGR的高分辨率结构 并鉴定PPGPP与MGLA-SSPA结合的抑制剂。特定目标2：确定弗朗西斯拉的结构 Cryo-Em的RNAP复合物。这些研究的成功完成将揭示一个新的范式 转录调节并使新型抗素养病毒治疗的合理设计。