Molecular elucidation of the Francisella tularensis virulence mechanism

图拉氏方济氏菌(Francisella tularensis，Ft)是图拉热病的病原，是最具传染性的细菌病原体之一。 为人所知。由于其高度传染性和易于气雾化，它已被列为A类生物武器 美国政府。图拉热症的发病率和死亡率是相当高的，鉴于其极端的传染性， 图拉热症的大规模爆发很容易压倒即使是美国最大的医疗机构的能力 中锋。FT毒力需要从染色体编码的Ft致病岛(FPI)表达的基因。 这些基因的表达被Ft调节剂的组合激活：严格的饥饿蛋白 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)， 它们在Ft感染过程中表达。MglA和PigR是Ft独有的，而SSPA蛋白在 多种细菌。然而，Ft SSPA是不寻常的，因为它不是均二聚体，而是异二聚体 和Mgla一起。PigR是一种DNA结合蛋白，其预测的DNA结合基序为有翼-螺旋-旋转-螺旋。 有趣的是，这种“警报”--鸟苷四磷酸(PpGpp)--也是Ft毒力所必需的。最近， 我们证明了这个警报直接与MglA-SSPA复合体结合。我们进一步证明了ppGpp结合 TO MglA-SSPA介导PigR与该杂二聚体的高亲和力结合。引人注目的是，我们的数据还显示， MglA-SSPA与Ft RNAPs70全酶构成相互作用，表明它代表一种毒力 专门化RNAP。鉴于Ft作为生物武器的极端毒性和潜在用途，目前有一个紧迫的问题 需要破译驱动其毒力的分子机制。这项提案的首要目标是 这些机制的分子解剖。我们的中心假设是，Ft采用了一种概念上的新形式 致病需要含有MglA-SSPA的毒力特异性RNAP。我们将测试我们的中央 推测并阐明控制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：充分描述MglA-SSPA与Ft的相互作用 RNAP。结构、生化和细胞研究将剖析MglA-A的毒力调节机制。 SSPA。具体目标2：对Ft RNAP(MglA-SSPA)-ppGpp-PigR进行结构和功能分析 复合体。将从结构上分析PigR介导的FPI激活背后的机制， 生化的和体内的。特定目标3：确定高分辨率(MglA-SSPA)-ppGpp-PigR晶体 与mglA-SSPA结合的ppGpp抑制剂的结构鉴定及结构的获得 复合体。这些目标的成功完成将揭示转录调控和 使新型抗链球菌毒力疗法的合理设计成为可能。

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.