Structural Elucidation of the Novel RNA Polymerase Underlying Francisella Tularensis Virulence

土拉热弗朗西丝菌（Francisellatularensis）是引起兔热病的病原菌之一， 知道的这种细菌可以很容易地雾化并用作生物武器。的发病率和死亡率 土拉菌病是重要的，考虑到弗朗西斯菌的感染能力， 甚至超过了美国最大的医疗中心。因此，弗朗西斯属被归类为 A类生化武器编码弗朗西斯菌致病岛（FPI）的基因， 是造成这种细菌毒性的原因严格饥饿蛋白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）介导这些蛋白的激活。 基因，因此对感染人类的弗朗西斯菌属物种的毒力至关重要。MglA和PigR SspA蛋白是Francisella特有的，而SspA蛋白存在于多种细菌中。弗朗西斯拉SspA， 然而，其不寻常之处在于它不同二聚化，而是作为与MglA的异二聚体起作用。PigR是一个 一种推测的DNA结合蛋白，具有预测的翼螺旋转角螺旋基序。SspA-MglA和PigR如何介导 FPI的激活是未知的，因为这些蛋白质用于感测的潜在分子机制也是未知的。 感染这项建议的首要目标是通过分子解剖这些机制， 人致病性土拉热弗朗西丝氏菌、土拉热弗朗西丝氏菌和holartica中这些毒力因子的研究 亚种。早期的研究表明，“警报素”，鸟苷四磷酸（ppGpp），是弗朗西斯菌的关键 毒性。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。 这种相互作用是通过解决MglA-SspA-ppGpp复合物结构来实现的。此外，我们发现ppGpp与 MglA-SspA介导PigR与该异二聚体的高亲和力结合。在本修订建议中，我们将 利用我们最近的发现来剖析弗朗西斯菌毒力调节系统的所有组件， 包括弗朗西斯氏菌RNA聚合酶（RNAP）。我们的中心假设是F. tularensis 采用了一种概念上新颖的毒力激活形式，涉及一种独特的RNAP， 活化复合物MglA-SspA作为核心成分。这得到了ChIP-seq研究和RNAP的支持 从Francisella细胞中纯化。我们将测试我们的中心假设，并完成提出的目标 通过两个具体目标。具体目的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 RNAP复合物通过cryo-EM。这些研究的成功完成将揭示一个新的模式， 转录调控，并能够合理设计新的抗弗朗西斯菌毒力治疗剂。

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.