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.

土拉热弗朗西斯菌 (Ft) 是兔热病的病原体，是最具传染性的细菌病原体之一 由于其高传染性和易于雾化，它被归类为A类生物武器。 美国政府指出，兔热病的发病率和死亡率很高，而且由于其传染性极强， 兔热病的大规模爆发很容易就会压垮美国最大的医疗机构的能力 Ft 毒力需要由染色体编码的 Ft 致病岛 (FPI) 表达的基因。 这些基因的表达由 Ft 调节因子的组合激活：严格饥饿蛋白 A (SspA)、巨噬细胞生长位点蛋白 A (MglA) 和致病岛基因调节因子 (PigR)， Ft 感染期间表达的 MglA 和 PigR 是 Ft 所特有的，而 SspA 蛋白则存在于 Ft 感染过程中。 然而，Ft SspA 是不寻常的，因为它不是同二聚体而是异二聚体。 PigR 是一种 DNA 结合蛋白，具有预测的翼状螺旋-转角-螺旋 DNA 结合基序。 有趣的是，“警报素”四磷酸鸟苷 (ppGpp) 最近也是 Ft 毒力所必需的。 我们表明该警报酮直接与 MglA-SspA 复合物结合。我们进一步表明 ppGpp 结合。 MglA-SspA 介导 PigR 与该异二聚体的高亲和力结合。 MglA-SspA 与 Ft RNAPs70 全酶发生组成型相互作用，表明它具有毒力 鉴于 Ft 的极端毒力和作为生物武器的潜在用途，迫切需要开发专门的 RNAP。 需要破译驱动其毒力的分子机制。该提案的首要目标是 我们的中心假设是 Ft 采用了一种概念上新颖的形式。 需要包含 MglA-SspA 的毒力特异性 RNAP 的发病机制我们将测试我们的中枢。 假设并描述了通过控制 Ft 毒力基因激活的分子机制 完成三个具体目标： 具体目标 1：充分表征 MglA-SspA 与 Ft 的相互作用 RNAP 的结构、生化和细胞研究将剖析 MglA- 的毒力调节机制。 具体目标2：进行Ft RNAP(MglA-SspA)-ppGpp-PigR的结构和功能分析 将对 PigR 介导的 FPI 激活背后的机制进行结构分析， 具体目标 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 这些目标的成功完成将揭示转录调控和转录调控的新范例。 能够合理设计新型抗弗朗西斯菌毒力疗法。