Assembly of bacterial cytoplasmic protein complexes measured by LRET

LRET 测量细菌细胞质蛋白复合物的组装

• 批准号: 8358552
• 负责人: ROBERT D RENTHAL
• 金额: $ 17.34万
• 依托单位: UNIVERSITY OF TEXAS SAN ANTONIO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-22 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8358552
• 关键词: Actins; Arsenicals; Bacteria; Binding; Cell Cycle; Cell division; Cells; Cytoplasm; Cytoplasmic Protein; Diffusion; Energy Transfer; Enterobacteriaceae; Escherichia coli; Ethanol; Exposure to; Face; Fiber; Filament; Fluorescein; Future; Glycols; Infection; Ions; Lanthanoid Series Elements; Length; Life; Link; Measures; Metabolic; Modeling; Molecular Structure; Organelles; Pathogenicity; Propane; Proteins; Proteus; Reaction; Relative (related person); Research; Role; Salmonella enterica; Shapes; Signal Transduction; Site; Structure; System; Terbium; Testing; Time; Toxic effect; Toxin; Vibrio cholerae; Virulence; expectation; in vivo; luminescence resonance energy transfer; pathogen; protein complex; research study; retinal rods; uptake

DESCRIPTION (provided by applicant): Bacterial cytoplasm contains remarkable ordered structures that are linked to pathogenicity and virulence. Actin-like filaments, made of the protei MreB, span the length of bacterial rods. Organelle-like bacterial microcompartments (BMCs), bounded by geometric protein shells, sequester volatile or toxic reaction intermediates. Cogwheel shaped tubules in many gram-negative pathogens help secrete toxins into targeted cells. Only limited information is available about the assembly and disassembly of these structures in vivo. Luminescence resonance energy transfer (LRET) will be used to test proposed models for actin-like filament assembly and bacterial microcompartment and tubule structure and dynamics. Recently it was discovered that Escherichia coli takes up luminescent terbium ions under conditions where these ions bind to genetically encoded sites without toxicity to the cells. The proposed experiments will exploit this finding to study molecular structures in live cells. MreB and the microcompartment shell proteins EutL and EutM will be tagged with tetraCys sequences, which will subsequently bind fluorescein arsenical to generate LRET acceptors. Terbium will be coordinated by a lanthanide-binding tag (LBT) on a cytoplasmic protein that will serve as the LRET donor. Because of the long excited state lifetimes of lanthanides, LRET is sensitive to the diffusion rates of donor and acceptor molecules. When the acceptors are released from ordered structures into the cytoplasm, a large increase in LRET should be detected, and when the acceptors are removed from the cytoplasm into ordered structures, a large decrease in LRET should be detected. This will provide spectroscopic signals of structure assembly and disassembly that can be followed during cell division and metabolic induction. Terbium uptake will also be measured in Salmonella enterica and Vibrio cholerae. If terbium uptake similar to E. coli is observed, studies will also be done on assembly of S. enterica Pdu microcompartments and V. cholerae type VI secretory system tubules. The results of these experiments will provide fundamental information about how bacterial actin-like filaments, microcompartments, and tubules are assembled in live cells. This information will aid future targeting of these structures to weaken the pathogenic effects of enterobacteria. PUBLIC HEALTH RELEVANCE: Some bacteria that live in the gut can cause severe infections, and recently discovered fibers, tubules and compartments inside these bacteria are linked to infectivity. The proposed research will study the assembly of the fibers, tubules and compartments, with the expectation that this information can be applied to new treatments for infection.

描述（由申请人提供）：细菌细胞质含有与致病性和毒力相关的显著有序结构。由蛋白质MreB组成的肌动蛋白样细丝横跨细菌棒的长度。细胞器样细菌微室（BMCs），由几何蛋白质外壳包围，隔离挥发性或有毒的反应中间体。许多革兰氏阴性病原体中的齿轮状小管有助于将毒素分泌到目标细胞中。关于这些结构在体内的组装和拆卸的信息有限。发光共振能量转移（LRET）将用于测试肌动蛋白样丝组装和细菌微室和微管结构和动力学的拟议模型。最近发现，大肠杆菌吸收发光的铽离子，条件是这些离子结合到基因编码的位点上，而对细胞没有毒性。拟议的实验将利用这一发现来研究活细胞的分子结构。MreB和微室壳蛋白EutL和EutM将用tetraCys序列标记，这些序列随后将结合荧光素砷产生LRET受体。铽将通过作为LRET供体的细胞质蛋白上的镧系结合标签（LBT）进行协调。由于镧系元素激发态寿命长，LRET对供体和受体分子的扩散速率敏感。当受体从有序结构释放到细胞质中时，应检测到LRET的大量增加，当受体从细胞质中移除到有序结构中时，应检测到LRET的大量减少。这将为细胞分裂和代谢诱导过程提供结构组装和拆卸的光谱信号。也将在肠沙门氏菌和霍乱弧菌中测量铽的摄取。如果观察到与大肠杆菌相似的铽摄取，还将研究肠球菌Pdu微室和霍乱弧菌VI型分泌系统小管的组装。这些实验的结果将提供关于细菌肌动蛋白样细丝、微室和小管如何在活细胞中组装的基本信息。这一信息将有助于未来针对这些结构，以削弱肠杆菌的致病作用。