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

基本信息

批准号：
10089396
负责人：
RICHARD GERALD BRENNAN
金额：
$ 19.48万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10089396
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

项目摘要

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.

土拉热弗朗西斯菌是土拉热病的病原体，是最具传染性的细菌病原体之一已知这种细菌可以容易地雾化并用作生物武器。土拉菌病很严重，鉴于弗朗西斯菌的传染能力，很容易发生大规模爆发弗朗西斯拉的能力甚至超过了美国最大的医疗中心的测试能力，被列为一类。美国政府的 A 类生物武器，在弗朗西斯菌致病岛 (FPI) 上编码的基因，该细菌的毒力由严格饥饿蛋白 A (SspA)（巨噬细胞）负责。生长位点蛋白 A (MglA) 和致病岛基因调节因子 (PigR) 介导这些基因的激活基因，因此对于感染人类的弗朗西斯菌属物种的毒力至关重要。是弗朗西斯菌所特有的，而弗朗西斯菌 SspA 蛋白存在于多种细菌中。然而，其不同寻常之处在于它不形成同二聚体，而是与 MglA 形成异二聚体。具有预测的翼状螺旋-转角-螺旋基序的假定 DNA 结合蛋白 SspA-MglA 和 PigR 如何介导。 FPI 激活尚不清楚，这些蛋白质用于感知的潜在分子机制也是未知的该提案的总体目标是通过分子解剖这些机制。人类致病性土拉弗朗西斯菌和霍拉蒂卡毒力因子的研究早期研究表明“警报素”、四磷酸鸟苷 (ppGpp) 是弗朗西斯菌的关键。我们最近证明 ppGpp 直接与 MglA-SspA 结合，并揭示了 ppGpp 的分子细节。通过解析 MglA-SspA-ppGpp 复合物结构，我们进一步证明了 ppGpp 的结合。 MglA-SspA 介导 PigR 与该异二聚体的高亲和力结合。利用我们最近的发现来剖析弗朗西斯菌毒力监管系统的所有组成部分，其中最重要的是弗朗西斯菌 RNA 聚合酶 (RNAP)，我们的中心假设是土拉弗朗西斯菌 (F. tularensis)。采用概念上新颖的毒力激活形式，一种包含毒力的独特RNAP 激活复合物 MglA-SspA 作为核心成分这一点得到了 ChIP-seq 研究和 RNAP 的支持。我们将测试我们的中心假设并完成拟议的目标。通过两个具体目标具体目标 1：阐明 (MglA-SspA)-ppGpp-PigR 的高分辨率结构。并鉴定 ppGpp 与 MglA-SspA 结合的抑制剂具体目标 2：确定弗朗西斯菌的结构。这些研究的成功完成将揭示 RNAP 复合物的新范例。转录调控并能够合理设计新型抗弗朗西斯菌毒力疗法。