Assembly of bacterial cytoplasmic protein complexes measured by LRET

描述(申请人提供)：细菌细胞质含有显著的有序结构，与致病性和致病力有关。肌动蛋白样细丝由Protei MreB组成，横跨细菌棒的长度。细胞器样细菌微室(BMC)，被几何蛋白质壳包围，隔离挥发性或有毒的反应中间产物。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靶细胞。关于这些结构在体内的组装和拆解，只有有限的信息可用。发光共振能量转移(LRET)将用于测试所提出的肌动蛋白样细丝组装和细菌微室和微管的结构和动力学模型。最近，人们发现，大肠杆菌在与基因编码的位置结合而不会对细胞产生毒性的条件下，会吸收发光的Tb离子。拟议中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将被标记有四个半胱氨酸序列，这些序列随后将与荧光素砷结合产生LRET受体。Tb将通过细胞质蛋白上的稀土元素结合标签(LBT)进行协调，该蛋白将作为LRET的供体。由于镧系元素的激发态寿命较长，LRET对施主和受主分子的扩散速率非常敏感。当受体从有序结构释放到细胞质中时，应该检测到LRET的大幅增加，当受体从细胞质中移除到有序结构中时，应该检测到LRET的大幅下降。这将提供在细胞分裂和代谢诱导过程中可以遵循的结构组装和分解的光谱信号。还将测量肠沙门氏菌和霍乱弧菌对Tb的吸收。如果观察到类似于大肠杆菌的Tb摄取，还将研究肠杆菌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有关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。这些信息将有助于未来针对这些结构来减弱肠道细菌的致病作用。 与公共卫生相关：一些生活在肠道中的细菌可导致严重感染，最近发现这些细菌内的纤维、小管和隔间与传染性有关。这项拟议的研究将研究纤维、小管和隔间的组装，期望这些信息可以应用于新的感染治疗方法。

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.