Protein Transport Across Membrane by Bacterial Pathogens

细菌病原体跨膜蛋白质运输

基本信息

批准号：
10711479
负责人：
Clarissa Lynn Durie
金额：
$ 38.08万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-07 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10711479
关键词：
Antibiotic Resistance Bacteria Bacterial Infections Binding Biochemical Biological Biological Assay Biological Models Biophysics Carrier Proteins Cell secretion Cells Complement Complex Coupled Cryoelectron Microscopy Defect Development Disease Engineering Environment Enzyme Kinetics Eukaryotic Cell Family Foundations Gene Delivery Genetic Hydrolysis Immune system In Vitro Infection Infection prevention Kinetics Label Legionella pneumophila Legionnaires&apos Disease Membrane Molecular Motor Nucleic Acids Nutrient Organelles Pathogenesis Peptide Signal Sequences Pharmaceutical Preparations Process Protein translocation Proteins Reaction Research Project Summaries Resolution Scientist Structure System Testing Therapeutic Thermodynamics Type IV Secretion System Pathway Virulence Virulence Factors Work biophysical analysis design experimental study extracellular fighting genetic approach insight macromolecule new technology next generation pathogenic bacteria protein transport public health relevance resistance gene response targeted delivery therapeutic protein tool trafficking

项目摘要

Project Summary This research seeks to understand the fundamental process of protein translocation across membrane barriers in bacteria. To establish an infection or exchange antibiotic resistance genes, bacteria must transport macromolecules including protein across multiple membrane barriers: their own and the host cell’s. In response to the universal requirement for macromolecule export, bacteria have evolved elaborate machineries called secretion systems that use energy to move macromolecules from the bacterial cell out into the extracellular milieu or directly into a host cell. In this proposal, I focus on the Type IV secretion system (T4SS). This family of secretion systems is unique in that there are T4SSs that can transport nucleic acid and/or protein cargo. As an important example of a T4SS, I will first investigate the defect in organelle trafficking / intracellular multiplication (Dot/Icm) T4SS in Legionella pneumophila. This system is essential for pathogenesis, which can result in the potentially fatal pneumonia Legionnaires’ Disease. The Dot/Icm T4SS is composed of 30 proteins in different copy numbers. It secretes over 300 protein substrates in order to evade the host cell’s immune system and scavenge nutrients. This represents a much larger repertoire of substrates than observed in other secretion systems that transport proteins out of the bacterial cell. Thus, the Dot/Icm T4SS is an ideal model system for determining how these numerous substrates are engaged and transported. Protein transport by T4SSs has traditionally been studied using cell-based assays. The cellular environment, however, does not allow for precise control and manipulation of reaction conditions. The field needs rigorous biophysical assays with which to dissect the molecular mechanism of protein translocation. I propose to combine determination of high-resolution structures of the Dot/Icm T4SS by cryoEM and thermodynamics and enzyme kinetics studies of the system. These approaches will complement traditional genetic and cell biological strategies and will lead to mechanistic insights into how this secretion system transports protein. For example, transient state kinetics experiments observing the ATP-dependent translocation of a fluorescently labeled substrate protein will answer questions such as “which signal sequences are recognized by which motor protein(s),” “are protein substrates unfolded during transport,” and “which kinetic steps are coupled to ATP binding and hydrolysis?” This approach to investigating complex cellular machinery by integrating biochemical, biophysical, structural, and genetic approaches will shed new light on the fundamental process of translocation across multiple membranes, an important feature of bacterial pathogenesis. While this work aims to understand the fundamental mechanism of protein translocation, our findings could lay the foundation for scientists to develop anti-virulence drugs, the next generation of tools fighting bacterial disease, and to engineer the targeted delivery of gene and protein therapeutics to eukaryotic cells by secretion systems.

项目概要这项研究旨在了解蛋白质跨膜易位的基本过程为了建立感染或交换抗生素抗性基因，细菌必须转运。大分子，包括跨越多个膜屏障的蛋白质：它们自己的和宿主细胞的。为了满足大分子输出的普遍要求，细菌进化出了复杂的称为分泌系统的机器，利用能量将大分子从细菌细胞转移到细胞外环境或直接进入宿主细胞在本提案中，我重点关注 IV 型分泌系统。 (T4SS) 该分泌系统家族的独特之处在于存在可以运输核酸的 T4SS。作为 T4SS 的一个重要例子，我将首先研究细胞器的缺陷。嗜肺军团菌中的运输/细胞内增殖 (Dot/Icm) T4SS 该系统对于嗜肺军团菌至关重要。发病机制，可能导致潜在致命的肺炎军团病 Dot/Icm T4SS。它由 30 种不同拷贝数的蛋白质组成，为了逃避而分泌 300 多种蛋白质底物。宿主细胞的免疫系统和清除营养物质，这代表了更大的底物库。比在将蛋白质转运出细菌细胞的其他分泌系统中观察到的要多，因此，Dot/Icm。 T4SS 是一个理想的模型系统，用于确定这些众多基材如何接合和运输。传统上，T4SS 的蛋白质转运是通过细胞环境进行研究的。然而，不允许对反应条件进行精确控制和操纵，该领域需要严格的条件。我建议用生物物理分析来剖析蛋白质易位的分子机制。通过冷冻电镜结合确定 Dot/Icm T4SS 的高分辨率结构和这些方法将补充系统的热力学和酶动力学研究。传统的遗传和细胞生物学策略，并将导致对这种分泌如何进行机制的深入了解例如，观察 ATP 依赖性的瞬态动力学实验。荧光标记的底物蛋白的易位将回答诸如“哪个信号序列由哪些运动蛋白识别”，“是在运输过程中展开的蛋白质底物”，以及 “哪些动力学步骤与 ATP 结合和水解相关？” 这种方法通过整合生物化学、生物物理、结构、遗传方法将为跨多个易位的基本过程提供新的线索膜，细菌发病机制的一个重要特征，而这项工作旨在了解蛋白质易位的基本机制，我们的发现可以为科学家开发奠定基础抗毒药物，对抗细菌性疾病的下一代工具，并设计靶向药物通过分泌系统将基因和蛋白质治疗剂递送至真核细胞。