Structural studies of virulence activation in Francisella tularensis

项目摘要/摘要 图拉热症的病原体是图拉氏菌，是已知的最具传染性的病原体之一，是一种 潜在的生物武器。弗朗西斯氏菌的毒力来源于一种称为弗朗西斯氏菌致病性的基因簇 岛(FPI)，其表达受一组独特的转录调控因子控制。MglA、SSPA和 PigR与应激信号四磷酸鸟苷或ppGpp合作，激活FPI处的转录。 然而，这些因子用来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可能是弗朗西塞拉RNA聚合酶(RNAP)MglA的一个完整的亚基 和SSPA形成具有结合ppGpp的开放空腔的异二聚体复合体，而PigR具有预测的 有翼螺旋-旋转-螺旋基序和非结构N-和C-末端与MglA-SSPA相互作用依赖于ppGpp 举止。基于这一数据，我们的中心假设是，fpi的毒力激活是由一种新的 MglA-SSPA是RNAP的一个亚单位，PigR从(MglA-SSPA)-ppGpp到DNA到DNA到 加强转录。这项提案的目标是揭示MglA-SSPA使用的交互机制 RNAP和PigR用来结合DNA和(MglA-SSPA)-ppGpp。这项工作将通过 完成两个具体目标。首先，我将解决一个高分辨率(MglA-SSPA)-PigR结构来辅助 基于结构的药物设计。在这个目标的第二部分，我将筛选一个通过 在Air Wise，Inc.的Silico筛选中，为了我的第二个目标，我提议利用单粒子低温EM来解决 多个Francisella RNAP复合体的结构。我将对这些结构研究进行后续的功能性研究 测试我们的基于结构的假说。我们期望这项工作将导致对 这种高度传染性的病原体的毒力激活机制，以及重要的是，我们的结构 将为合理的药物设计提供Francisella独有的新靶点。 我的培训计划的一个重要部分是获得X射线结晶学和单粒子冷冻-EM方面的专业知识。我 建议通过课程作业、我的赞助人舒马赫博士和合作者舒马赫博士的培训来做到这一点。 Bartesaghi，他们是这些领域的专家。我还解释了我将如何加强我在微生物学方面的背景， 学会领导一个研究项目，成为一名优秀的导师和合作者，并在我的科学研究基础上不断进步 沟通技巧。培训计划将使我具备完成培训所需的知识和技能。 提出研究并实现我成为一名独立研究人员的长期目标 结构生物学。这项研究将在舒马赫实验室进行，作为 杜克大学的生物化学，该校在培训杰出研究人员方面有着丰富的历史，并将提供 出色的环境和资源，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.