Control of virulence gene expression in Francisella tularensis

描述(申请人提供)：图拉氏方济氏菌是图拉热症的病原体，是目前已知的最具传染性的细菌病原体之一，也是NIAID A类优先病原体，因为它有可能用作生物武器。在图拉氏丝虫中，SSPA蛋白家族成员MglA和SSPA在调节毒力基因的表达方面发挥关键作用，这些基因是巨噬细胞生长和生存所必需的，也是生物体致病生活方式的关键。MglA和SSPA形成一个复合体，与RNA聚合酶(RNAP)结合，通过一种尚不清楚的机制积极控制毒力基因的表达。我们发现，在图拉氏杆菌活疫苗株中，MglA-SSPA复合体与小分子ppGpp和一种可能的DNA结合蛋白(我们称之为PigR)协同工作，控制一组共同基因的表达。我们已经获得了PigR直接与MglA-SSPA复合体相互作用的证据，而ppGpp通过促进这种相互作用发挥作用。虽然ppGpp在许多其他病原体中起着关键的调节作用，但我们的发现表明，在Francisella中，ppGpp可能通过一种新的机制来控制基因的表达--通过促进转录激活因子与SSPA蛋白家族成员形成的RNAP相关复合体之间的相互作用。在本提案的目标1中，我们将使用生化和遗传方法相结合的方法来确定MglA、SSPA、PigR和ppGpp如何发挥其转录效应。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与MglA-SSPA复合体之间的蛋白质-蛋白质相互作用是否是这三种蛋白质调节活性的关键，并确定ppGpp如何调节PigR与MglA-SSPA复合体之间的相互作用。我们最近的发现表明，TrmE蛋白参与了MglA/SSPA调控基因的调控。TrmE是一种假定的GTP酶，已被证明影响其他生物体的蛋白质合成。我们假设TrmE通过影响细胞内ppGpp、MglA、SSPA、PigR或其他调节因子的浓度而对基因表达产生影响。在目标2中，我们将采用遗传和生化方法来确定TrmE如何影响MglA/SSPA调节基因的表达。这项拟议的工作有望揭示一种重要的细胞内病原体如何调控宿主中生存所需的基因的表达，并对ppGpp和SSPA家族成员如何调控其他病原菌的基因表达具有影响。 公共卫生相关性：图拉氏方济氏菌是图拉热症的病原体，是一种细胞内病原体，也是一种潜在的生物武器。人们对弗朗西斯氏菌细胞内生长所需的基因是如何调控的知之甚少。我们建议确定一个小分子ppGpp如何与RNA聚合酶相关的MglA-SSPA复合体以及一个名为PigR的DNA结合蛋白协同工作，以积极控制使弗朗西斯菌能够在细胞内生长的基因的表达。PpGpp的活性与MglA/SSPA类调控因子之间的联系以前还没有被认识到，并且可能延伸到其他病原体，在这些调控因子在调节与宿主的相互作用中发挥重要作用。拟议中的实验有望揭示一种重要的细胞内病原体如何调节致病所需基因的表达。我们的工作对可能预防图拉热症的疫苗的开发具有重要意义。

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.