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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：
10066958
负责人：
Brady A Travis
金额：
$ 3.93万
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 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10066958
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 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.

项目总结/文摘