Control of virulence gene expression in Francisella tularensis

描述（由申请方提供）：土拉热弗朗西斯菌是土拉菌病的病原体，是目前已知的最具传染性的细菌病原体之一，由于其可用作生物武器，因此是NIAID A类优先病原体。in F. Tularensis中，SspA蛋白家族成员MglA和SspA在调节毒力基因的表达中起关键作用，所述毒力基因对于巨噬细胞内生长和存活是必需的，并且是生物体致病生活方式的关键。MglA和SspA形成与RNA聚合酶（RNAP）缔合的复合物，以通过尚不清楚的机制积极控制毒力基因的表达。我们发现，在活疫苗株F。在tularensis中，MglA-SspA复合物与小分子ppGpp和我们命名为PigR的推定DNA结合蛋白协同作用，以控制一组共同基因的表达。我们已经获得了PigR直接与MglA-SspA复合物相互作用的证据，并且ppGpp通过促进这种相互作用发挥其作用。虽然ppGpp在许多其他病原体中起着关键的调节作用，我们的研究结果表明，在弗朗西斯菌ppGpp可能控制基因表达通过一种新的机制-通过促进转录激活因子和RNAP相关的复合物形成的SspA蛋白家族的成员之间的相互作用。在本提案的目标1中，我们将使用生物化学和遗传方法的组合来确定MglA、SspA、PigR和ppGpp如何发挥其转录作用。特别是，我们建议确定PigR是否通过与DNA接触发挥作用，明确测试PigR和MglA-SspA复合物之间的蛋白质-蛋白质相互作用是否是这三种蛋白质的调节活性的关键，并确定ppGpp如何调节PigR和MglA-SspA复合物之间的相互作用。 我们最近的研究结果表明TrmE蛋白参与了MglA/SspA调控基因的调控。TrmE是一种推定的GTdR，已被证明会影响其他生物体中的蛋白质合成。我们假设TrmE通过影响ppGpp、MglA、SspA、PigR或另一种调节剂的细胞内浓度来对基因表达发挥其作用。在目标2中，我们将采取遗传和生物化学方法来确定TrmE如何影响MglA/SspA调节基因的表达。 这项工作有望揭示一种重要的细胞内病原体如何调节宿主生存所需的基因表达，并对ppGpp和SspA家族成员如何调节其他病原菌的基因表达具有影响。 公共卫生相关性：土拉热弗朗西斯菌是土拉菌病的病原体，是一种细胞内病原体和潜在的生物武器。关于弗朗西斯菌细胞内生长所需的基因是如何调控的，我们知之甚少。我们建议，以确定如何一个小分子，ppGpp，协同工作的RNA聚合酶相关的MglA-SspA复合物，和一个假定的DNA结合蛋白命名PigR，积极控制基因的表达，使弗朗西斯菌细胞内生长。ppGpp和MglA/SspA类调节剂的活性之间的联系以前没有被认识到，并且可以延伸到其他病原体，其中这些调节剂在介导与宿主的相互作用中发挥重要作用。拟议的实验有望揭示一种重要的细胞内病原体如何调节引起疾病所需的基因表达。我们的工作对开发可能预防兔热病的疫苗具有意义。

DESCRIPTION (provided by applicant): Francisella tularensis, the aetiological agent of tularemia, is one of the most infectious bacterial pathogens currently known and a NIAID category A priority pathogen because of its potential for use as a biological weapon. In F. tularensis, the SspA protein family members MglA and SspA play a critical role in regulating the expression of virulence genes that are essential for intramacrophage growth and survival, and are key to the organisms pathogenic lifestyle. MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes by a mechanism that is not understood. We have found that in the live vaccine strain of F. tularensis, the MglA-SspA complex works in concert with the small molecule ppGpp, and a putative DNA-binding protein we have named PigR, to control the expression of a common set of genes. We have obtained evidence that PigR interacts directly with the MglA-SspA complex, and that ppGpp exerts its effect by promoting this interaction. Although ppGpp plays a critical regulatory role in many other pathogens, our findings suggest that in Francisella ppGpp may control gene expression through a novel mechanism-by promoting the interaction between a transcription activator and an RNAP-associated complex formed by members of the SspA protein family. In Aim 1 of this proposal we will use a combination of biochemical and genetic approaches to determine how MglA, SspA, PigR and ppGpp exert their transcriptional effects. In particular, we propose to determine whether PigR functions through contact with the DNA, to explicitly test whether the protein-protein interaction between PigR and the MglA-SspA complex is key to the regulatory activities of these three proteins, and to determine how ppGpp modulates the interaction between PigR and the MglA-SspA complex. Our recent findings implicate the TrmE protein in the control of MglA/SspA-regulated genes. TrmE is a putative GTPase that has been shown to influence protein synthesis in other organisms. We hypothesize that TrmE exerts its effects on gene expression through an effect on the intracellular concentration of ppGpp, MglA, SspA, PigR, or another regulator. In Aim 2, we will take both genetic and biochemical approaches to determine how TrmE influences the expression of MglA/SspA-regulated genes. The proposed work is expected to reveal how an important intracellular pathogen regulates the expression of genes required for survival in the host, and has implications for how ppGpp and SspA family members regulate gene expression in other pathogenic bacteria. PUBLIC HEALTH RELEVANCE: Francisella tularensis, the causative agent of tularemia, is an intracellular pathogen and a potential bioweapon. Little is known about how genes required for the intracellular growth of Francisella are regulated. We propose to determine how a small molecule, ppGpp, works in concert with the RNA polymerase-associated MglA-SspA complex, and a putative DNA-binding protein named PigR, to positively control the expression of genes that enable Francisella to grow intracellularly. A connection between the activities of ppGpp and regulators of the MglA/SspA class has not previously been appreciated, and may extend to other pathogens where these regulators play important roles in mediating interactions with the host. The proposed experiments are expected to reveal how an important intracellular pathogen regulates the expression of genes that are required to cause disease. Our work has implications for the development of vaccines that may protect against tularemia.