Molecular elucidation of the Francisella tularensis virulence mechanism

土拉热弗朗西丝菌（Francisellatularensis，Ft）是土拉菌病的病原菌之一 知道的由于其高传染性和易于雾化，它已被列为A类生物武器， 美国政府。土拉菌病的发病率和死亡率很高，鉴于其极端的传染性， 严重的土拉菌病爆发将很容易超过美国最大的医疗机构的能力， 中心. 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）， 在Ft感染期间表达。MglA和PigR是Ft所特有的，而SspA蛋白在Ft中被发现。 多种细菌然而，Ft SspA是不寻常的，因为它不同源二聚化而是异源二聚化 关于MGA PigR是一种DNA结合蛋白，具有预测的翼螺旋-转角螺旋DNA结合基序。 有趣的是，“报警素”，鸟苷四磷酸（ppGpp），也是必要的Ft毒力。最近， 我们发现这种alarmone直接结合到MglA-SspA复合物上。我们进一步表明，ppGpp结合 MglA-SspA介导PigR与该异源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全酶组成型相互作用，表明其代表毒力 专业RNAP考虑到Ft作为生物武器的极端毒性和潜在用途， 需要破译其致病的分子机制。本提案的总体目标是 这些机制的分子解剖。我们的中心假设是，Ft采用了一种概念上新颖的形式， 的发病机制需要一个毒力特异性RNAP含有MglA-SspA。我们将测试我们的中央 假设，并描绘控制Ft毒力基因激活的分子机制， 具体目标1：充分表征MglA-SspA与Ft的相互作用 RNAP。结构、生物化学和细胞研究将剖析MglA的毒力调节机制， SspA。具体目的2：进行Ft RNAP（MglA-SspA）-ppGpp-PigR的结构和功能分析 配合物将从结构上分析PigR介导的FPI激活背后的机制， 生物化学和体内。具体目的3：确定高分辨率（MglA-SspA）-ppGpp-PigR晶体 结构，鉴定ppGpp与MglA-SspA结合抑制剂，并获得MglA-SspA抑制剂的结构 配合物这些目标的成功完成将揭示转录调控的新范式， 能够合理设计新型抗弗朗西斯菌毒力治疗剂。

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.