Structural studies of virulence activation in Francisella tularensis

项目概要/摘要 土拉菌病的病原体，土拉弗朗西斯菌，是已知的最具感染性的病原体之一， 潜在的生化武器弗朗西斯菌毒力源于一个被称为弗朗西斯菌致病性的基因簇 岛（FPI），其表达受一组独特的转录调节因子控制。MglA、SspA和 PigR与应激信号鸟苷四磷酸或ppGpp协作以激活FPI处的转录。 然而，这些因子用于驱动毒力激活的分子机制尚不清楚。在最近的研究中， 我们已经证明MglA和SspA可能是Francisella RNA聚合酶（RNAP）的一个完整亚基， 和SspA形成具有结合ppGpp和PigR的开放腔的异二聚体复合物，其具有预测的 有翼螺旋-转角-螺旋基序和非结构化的N-和C-末端，与MglA-SspA以ppGpp依赖性相互作用。 方式基于这些数据，我们的中心假设是，FPI的毒力激活是由一种新的 其中MglA-SspA是RNAP的亚基并且PigR将（MglA-SspA）-ppGpp桥连到DNA以 增强转录。该提案的目标是揭示MglA-SspA用于与 RNAP和PigR用于结合DNA和（MglA-SspA）-ppGpp。这项工作将通过 实现两个具体目标。首先，我将求解一个高分辨率（MglA-SspA）-PigR结构， 基于结构的药物设计。在这个目标的第二部分，我将筛选一个小的抑制剂库，通过 Atomwise，Inc.的计算机模拟筛选对于我的第二个目标，我建议利用单粒子冷冻EM来解决 多个Francisella RNAP复合物的结构。我将继续进行这些结构研究， 来检验我们的结构假设。我们希望这项工作将有助于了解 这种高度传染性病原体的毒力激活机制，重要的是，我们的结构 将为弗朗西斯菌提供独特的新靶点，用于合理的药物设计。 我的培训计划的一个重要部分是获得X射线晶体学和单粒子冷冻EM的专业知识。我 我建议通过课程作业，我的赞助商，舒马赫博士和合作者，博士培训。 Bartesaghi是这些领域的专家。我还解释了我将如何加强我的微生物学背景， 学习领导一个研究项目，成为一个优秀的导师和合作者，并提高我的科学 沟通能力培训计划将使我具备完成 我提出的研究，并实现我的长期目标，成为一个独立的研究人员在该领域的 结构生物学这项研究将在舒马赫实验室进行，作为该部门的一部分。 杜克大学的生物化学专业在培养杰出的研究人员方面有着丰富的历史， 优秀的环境和资源，使我能够实现我的目标。

PROJECT SUMMARY/ABSTRACT The etiological agent of tularemia, Francisella tularensis, is one of the most infectious pathogens known and a potential bioweapon. Francisella virulence stems from a gene cluster known as the Francisella pathogenicity island (FPI) whose expression is under the control of a unique set of transcriptional regulators. MglA, SspA, and PigR collaborate with the stress signal, guanosine tetraphosphate, or ppGpp, to activate transcription at the FPI. However, the molecular mechanisms these factors use to drive virulence activation is unclear. In recent studies, we have shown that MglA and SspA may be an integral subunit of Francisella RNA polymerase (RNAP), MglA and SspA form a heterodimeric complex with an open cavity that binds ppGpp, and PigR, which has a predicted winged helix-turn-helix motif and unstructured N- and C-termini, interacts with MglA-SspA in a ppGpp-dependent manner. Based on this data, our central hypothesis is that virulence activation at the FPI occurs by a novel mechanism where MglA-SspA is a subunit of RNAP and PigR bridges from (MglA-SspA)-ppGpp to DNA to enhance transcription. The goal of this proposal is to uncover the mechanisms MglA-SspA uses to interact with RNAP and PigR uses to bind DNA and (MglA-SspA)-ppGpp. This work will be accomplished through the completion of two specific aims. First, I will solve a high-resolution (MglA-SspA)-PigR structure to aide in structure-based drug design. In the second part of this aim, I will screen a small library of inhibitors identified via in silico screening by Atomwise, Inc. For my second aim, I propose to utilize single-particle cryo-EM to solve structures of multiple Francisella RNAP complexes. I will follow up on these structural studies with functional assays to test our structure-based hypotheses. We expect that this work will lead to an understanding of the mechanisms underlying virulence activation in this highly infectious pathogen and, importantly, our structures will provide novel targets unique to Francisella to be used for rational drug design. A significant part of my training plan is to gain expertise in X-ray crystallography and single-particle cryo-EM. I propose to do this through coursework, training from my sponsor, Dr. Schumacher, and collaborator, Dr. Bartesaghi, who are experts in these fields. I also explain how I will strengthen my background in microbiology, learn to lead a research project, become an excellent mentor and collaborator, and improve upon my scientific communication skills. The training plan will equip me with the knowledge and skills needed to complete the proposed research and achieve my long-term goal of becoming an independent researcher in the field of structural biology. This research will be conducted in the Schumacher laboratory as part of the Department of Biochemistry at Duke University, which has a rich history of training remarkable investigators and will provide an outstanding environment and resources that will allow me to accomplish my goals.