Structural studies of virulence activation in Francisella tularensis

土拉弗朗西斯菌毒力激活的结构研究

基本信息

批准号：
10322359
负责人：
Brady A Travis
金额：
$ 3.35万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10322359
关键词：
Bacteria Binding Biochemical Biochemistry Biological Assay Biology Bioterrorism C-terminal Categories Cells Communication Complex Cryoelectron Microscopy Crystallization DNA DNA Binding DNA Structure DNA-Binding Proteins DNA-Directed DNA Polymerase DNA-Directed RNA Polymerase Data Development Disease Drug Design Environment Etiology Fluorescence Polarization Francisella Francisella tularensis Gene Cluster Gene Expression Genes Genetic Transcription Goals Growth Guanine Guanosine Tetraphosphate Helix-Turn-Helix Motifs Holoenzymes Homodimerization Inhalation Knowledge Laboratories Lead Learning Libraries Link Mediating Mentors Microbiology Molecular Multi-Drug Resistance Organism Pathogenicity Pathogenicity Island Polymerase Proteins Recording of previous events Regulator Genes Research Research Personnel Research Project Grants Resolution Resources Signal Transduction Starvation Stress Structure System Tail Testing Training Transcriptional Activation Transcriptional Regulation Tularemia United States Dept. of Health and Human Services Universities Virulence Winged Helix Work X-Ray Crystallography aerosolized base bioweapon combat design environmental stressor follow-up improved in silico inhibitor/antagonist insight interest macrophage novel novel therapeutics particle pathogen promoter recruit response screening skills small molecule stem structural biology transcription factor weapons

项目摘要

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.

项目概要/摘要土拉热病的病原体土拉弗朗西斯菌是已知最具传染性的病原体之一，也是一种已知的传染性最强的病原体。潜在的生物武器。弗朗西斯菌毒力源于称为弗朗西斯菌致病性的基因簇岛（FPI），其表达受到一组独特的转录调节因子的控制。 MglA、SspA 和 PigR 与应激信号、四磷酸鸟苷或 ppGpp 协作，激活 FPI 处的转录。然而，这些因素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 可能是弗朗西斯菌 RNA 聚合酶 (RNAP) 的一个完整亚基，MglA 和 SspA 形成具有开放腔的异二聚体复合物，该复合物结合 ppGpp 和 PigR，其具有预测的翼状螺旋-转角-螺旋基序和非结构化 N 端和 C 端，以 ppGpp 依赖性方式与 MglA-SspA 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 MglA-SspA 是 RNAP 的一个亚基，PigR 从 (MglA-SspA)-ppGpp 桥接到 DNA 的机制增强转录。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。这项工作将通过完成两个具体目标。首先，我将求解高分辨率 (MglA-SspA)-PigR 结构以帮助基于结构的药物设计。在这个目标的第二部分中，我将筛选一个小型抑制剂库，通过 Atomwise, Inc. 进行的计算机筛选。对于我的第二个目标，我建议利用单粒子冷冻电镜来解决多个弗朗西斯菌 RNAP 复合物的结构。我将用功能来跟进这些结构研究测试我们基于结构的假设。我们希望这项工作能够让人们更好地理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提供弗朗西斯拉特有的新靶标，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电镜方面的专业知识。我建议通过我的赞助商舒马赫博士和合作者舒马赫博士的课程和培训来做到这一点。 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，成为一名优秀的导师和合作者，并提高我的科学水平沟通技巧。培训计划将使我具备完成任务所需的知识和技能提出研究并实现我成为该领域的独立研究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，作为该部门的一部分杜克大学的生物化学学院拥有培养杰出研究人员的悠久历史，并将提供优越的环境和资源将使我能够实现我的目标。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structural Basis for Virulence Activation of Francisella tularensis.

DOI：
10.1016/j.molcel.2020.10.035
发表时间：
2021-01-07
期刊：
Molecular cell
影响因子：
16
作者：
Travis BA;Ramsey KM;Prezioso SM;Tallo T;Wandzilak JM;Hsu A;Borgnia M;Bartesaghi A;Dove SL;Brennan RG;Schumacher MA
通讯作者：
Schumacher MA

Diverse molecular mechanisms of transcription regulation by the bacterial alarmone ppGpp.