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）：细菌细胞质包含与致病性和毒力相关的显着有序结构。由蛋白质MREB制成的肌动蛋白样细丝横跨细菌棒的长度。细菌样细菌微室（BMC），受几何蛋白壳，隔离挥发性或有毒反应中间体的范围。许多革兰氏阴性病原体中的齿轮形小管有助于将毒素分泌到靶细胞中。只有有关这些结构在体内的组装和拆卸的有限信息。发光谐振能量转移（LRET）将用于测试肌动蛋白样细丝组件和细菌微型室以及小管结构和动力学的模型。最近发现，在这些离子与遗传编码的位点结合而没有毒性的条件下，大肠杆菌吸收发光的terbium离子。提出的实验将利用这一发现来研究活细胞中的分子结构。 MREB和MicroCompartment壳蛋白EUTL和EUTM将用Tetracys序列标记，随后将结合荧光素砷以生成Lret受体。 Terbium将通过胞质蛋白上的Lanthanide结合标签（LBT）协调，该蛋白将用作LRET供体。由于灯笼的激发态寿命长，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LER的大幅增加，当将受体从细胞质中移除到有序结构中时，应检测到LRET的大幅度减少。这将提供结构组装和拆卸的光谱信号，并在细胞分裂和代谢诱导过程中遵循。 Terbium摄取也将在肠沙门氏菌和弧菌霍乱中进行测量。如果观察到类似于大肠杆菌的Terbium摄取，还将对肠链球菌PDU微型营地和V.霍乱VI型分泌系统小管进行研究。这些实验的结果将提供有关细菌肌动蛋白样细丝，微型室和小管如何组装在活细胞中的基本信息。这些信息将有助于将来靶向这些结构，以削弱肠杆菌的致病作用。 公共卫生相关性：一些生活在肠道中的细菌可能引起严重的感染，最近发现这些细菌内部的纤维，小管和隔室与感染性有关。拟议的研究将研究纤维，小管和隔室的组装，并期望将此信息应用于新的感染治疗方法。