Structural Elucidation of the Novel RNA Polymerase Underlying Francisella Tularensis Virulence

图拉氏方济氏菌是图拉热症的病原体，是最具传染性的细菌病原体之一。 为人所知。这种细菌可以很容易地雾化并用作生物武器。该病的发病率和死亡率 图拉热症是严重的，考虑到弗朗西斯氏菌的感染能力，大爆发很容易 甚至压倒了美国最大的医疗中心的能力。因此，弗朗西塞拉被归类为 美国政府的A类生物武器。弗朗西塞氏菌致病岛(FPI)上编码的基因， 对这种细菌的毒力负有责任。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)介导这些基因的激活 基因，因此对感染人类的弗朗西斯氏菌的毒力是必不可少的。MglA和PigR 是弗朗西斯氏菌所特有的，而SSPA蛋白存在于多种细菌中。弗朗西塞拉SSPA， 然而，不同寻常的是，它不是均二聚体，而是与MglA一起作为异源二聚体发挥作用。PigR是一个 推测的DNA结合蛋白，具有预测的有翼-螺旋-旋转-螺旋基序。SSPA-MglA和PigR如何调节 Fpi的激活是未知的，这些蛋白质用来感知的潜在分子机制也是未知的。 感染。这个提议的首要目标是通过分子解剖这些机制 人致病性图拉氏方济氏菌和霍拉克氏杆菌毒力因子的研究 亚种。早期的研究表明，鸟苷-四磷酸鸟苷(PpGpp)是弗氏杆菌的关键成分 致命性。我们最近发现ppGpp直接与MglA-SSPA结合，并揭示了MglA-SSPA的分子细节 这种相互作用通过求解MglA-SSPA-ppGpp的复杂结构来实现。此外，我们还展示了ppGpp与 MglA-SSPA介导PigR与异源二聚体的高亲和力结合。在这份修订后的提案中，我们将 利用我们最近的发现来剖析弗朗西斯氏菌毒力监管系统的所有组成部分， 关键的是，包括弗朗西塞拉RNA聚合酶(RNAP)。我们的中心假设是图拉氏丝虫 采用一种概念上新颖的毒力激活形式，涉及包含毒力的独特RNAP 激活复杂的MglA-SSPA作为核心成分。这得到了CHIP-SEQ研究和RNAP的支持 从弗朗西塞拉细胞中提纯。我们将检验我们的中心假设，并完成所提出的目标 通过两个具体目标。特定目的1：阐明(MglA-SSPA)-ppGpp-PigR的高分辨结构 并鉴定与mglA-SSPA结合的ppGpp的抑制剂。具体目标2：确定方济各氏菌的结构 冷冻-EM法制备RNAP复合体。这些研究的成功完成将揭示一个新的范式 转录调控，并使合理设计新的抗链球菌毒力疗法。

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.